U.S. patent application number 17/519790 was filed with the patent office on 2022-02-24 for treatment method.
The applicant listed for this patent is Hoffmann-La Roche Inc.. Invention is credited to Marina BACAC, Stefan EVERS, Christian KLEIN, Pavel PISA, Eva ROSSMANN, Jose SARO, Pablo UMANA.
Application Number | 20220054635 17/519790 |
Document ID | / |
Family ID | |
Filed Date | 2022-02-24 |
United States Patent
Application |
20220054635 |
Kind Code |
A1 |
BACAC; Marina ; et
al. |
February 24, 2022 |
TREATMENT METHOD
Abstract
The present invention relates to methods of treating a disease,
and methods for reduction of the formation of anti-drug antibodies
(ADAs) in response to the administration of a therapeutic agent.
The invention further relates to methods of treating a disease,
particularly a B-cell proliferative disorder, and methods for
reduction of adverse effects in response to the administration of a
therapeutic agent, particularly a T-cell activating therapeutic
agent.
Inventors: |
BACAC; Marina; (Schlieren,
CH) ; EVERS; Stefan; (Schlieren, CH) ; KLEIN;
Christian; (Schlieren, CH) ; PISA; Pavel;
(Schlieren, CH) ; ROSSMANN; Eva; (Basel, CH)
; SARO; Jose; (Schlieren, CH) ; UMANA; Pablo;
(Schlieren, CH) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Hoffmann-La Roche Inc. |
Little Falls |
NJ |
US |
|
|
Appl. No.: |
17/519790 |
Filed: |
November 5, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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17240821 |
Apr 26, 2021 |
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17519790 |
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15371891 |
Dec 7, 2016 |
11013801 |
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17240821 |
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International
Class: |
A61K 39/395 20060101
A61K039/395; C07K 16/30 20060101 C07K016/30; C07K 16/28 20060101
C07K016/28 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 9, 2015 |
EP |
15198715.3 |
Jun 2, 2016 |
EP |
16172739.1 |
Oct 10, 2016 |
EP |
16193151.4 |
Claims
1. A Type II anti-CD20 antibody for use in a method of treating a
disease in a subject, the method comprising a treatment regimen
comprising (i) administration to the subject of the Type II
anti-CD20 antibody, and consecutively after a period of time (ii)
administration to the subject of a therapeutic agent, wherein the
period of time between the administration of the Type II anti-CD20
antibody and the administration of the therapeutic agent is
sufficient for reduction of the number of B-cells in the subject in
response to the administration of the Type II anti-CD20
antibody.
2. The Type II anti-CD20 antibody of claim 1, wherein the treatment
regimen effectively reduces the formation of anti-drug antibodies
(ADAs) in the subject in response to the administration of the
therapeutic agent as compared to a corresponding treatment regimen
without the administration of the anti-CD20 antibody.
3. The Type II anti-CD20 antibody of claim 1 or 2, wherein the
treatment regimen effectively reduces cytokine release associated
with the administration of the therapeutic agent in the subject as
compared to a corresponding treatment regimen without the
administration of the Type II anti-CD20 antibody.
4. The Type II anti-CD20 antibody of claim 3, wherein the
therapeutic agent is a T cell activating therapeutic agent.
5. A Type II anti-CD20 antibody for use in a method for (i)
reducing the formation of anti-drug antibodies (ADAs) against a
therapeutic agent in a subject and/or (ii) reducing cytokine
release associated with the administration a therapeutic agent,
particularly a T-cell activating therapeutic agent, in a subject,
comprising administration of the Type II anti-CD20 antibody to the
subject prior to administration of the therapeutic agent.
6. The Type II anti-CD20 antibody of claim 5, wherein the period of
time between the administration of the Type II anti-CD20 antibody
and administration of the therapeutic agent is sufficient for
reduction of the number of B-cells in the subject in response to
the administration of the Type II anti-CD20 antibody.
7. The Type II anti-CD20 antibody of any one of the preceding
claims, wherein the anti-CD20 antibody comprises a heavy chain
variable region comprising the heavy chain CDR (HCDR) 1 of SEQ ID
NO: 4, the HCDR2 of SEQ ID NO: 5, and the HCDR3 of SEQ ID NO: 6;
and a light chain variable region comprising the light chain CDR
(LCDR) 1 of SEQ ID NO: 7, the LCDR2 of SEQ ID NO: 8 and the LCDR3
of SEQ ID NO: 9.
8. The Type II anti-CD20 antibody of any one of the preceding
claims, wherein the anti-CD20 antibody comprises the heavy chain
variable region sequence of SEQ ID NO: 10 and the light chain
variable region sequence of SEQ ID NO: 11.
9. The Type II anti-CD20 antibody of any one of the preceding
claims, wherein the anti-CD20 antibody is an IgG antibody,
particularly an IgG.sub.1 antibody, and wherein at least about 40%
of the N-linked oligosaccharides in the Fc region of the anti-CD20
antibody are non-fucosylated.
10. The Type II anti-CD20 antibody of any one of the preceding
claims, wherein the anti-CD20 antibody is obinutuzumab.
11. The Type II anti-CD20 antibody of any one of the preceding
claims, wherein the therapeutic agent comprises a polypeptide.
12. The Type II anti-CD20 antibody of any one of the preceding
claims, wherein the therapeutic agent comprises an antibody.
13. The Type II anti-CD20 antibody of claim 12, wherein the
antibody comprised in the therapeutic agent specifically binds to
carcinoembryonic antigen (CEA).
14. The Type II anti-CD20 antibody of claim 13, wherein the
antibody comprised in the therapeutic agent comprises (i) a heavy
chain variable region comprising the heavy chain CDR (HCDR) 1 of
SEQ ID NO: 14, the HCDR2 of SEQ ID NO: 15, and the HCDR3 of SEQ ID
NO: 16; and a light chain variable region comprising the light
chain CDR (LCDR) 1 of SEQ ID NO: 17, the LCDR2 of SEQ ID NO: 18 and
the LCDR3 of SEQ ID NO: 19; or (ii) a heavy chain variable region
sequence of SEQ ID NO: 20 and a light chain variable region
sequence of SEQ ID NO: 21.
15. The Type II anti-CD20 antibody of claim 12, wherein the
antibody comprised in the therapeutic agent specifically binds to
CD3, particularly CD3.epsilon..
16. The Type II anti-CD20 antibody of claim 15, wherein the
antibody comprised in the therapeutic agent comprises (i) a heavy
chain variable region comprising the heavy chain CDR (HCDR) 1 of
SEQ ID NO: 32, the HCDR2 of SEQ ID NO: 33, and the HCDR3 of SEQ ID
NO: 34; and a light chain variable region comprising the light
chain CDR (LCDR) 1 of SEQ ID NO: 35, the LCDR2 of SEQ ID NO: 36 and
the LCDR3 of SEQ ID NO: 37; or (ii) a heavy chain variable region
sequence of SEQ ID NO: 38 and a light chain variable region
sequence of SEQ ID NO: 39.
17. The Type II anti-CD20 antibody of claim 12, wherein the
antibody comprised in the therapeutic agent specifically binds to
CD20.
18. The Type II anti-CD20 antibody of claim 15, wherein the
antibody comprised in the therapeutic agent comprises (i) a heavy
chain variable region comprising the heavy chain CDR (HCDR) 1 of
SEQ ID NO: 4, the HCDR2 of SEQ ID NO: 5, and the HCDR3 of SEQ ID
NO: 6; and a light chain variable region comprising the light chain
CDR (LCDR) 1 of SEQ ID NO: 7, the LCDR2 of SEQ ID NO: 8 and the
LCDR3 of SEQ ID NO: 9; or (ii) a heavy chain variable region
sequence of SEQ ID NO: 10 and a light chain variable region
sequence of SEQ ID NO: 11.
19. The Type II anti-CD20 antibody of any one of the preceding
claims, wherein the therapeutic agent comprises a cytokine,
particularly interleukin-2.
20. The Type II anti-CD20 antibody of claim 19, wherein the
cytokine is a mutant human IL-2 polypeptide comprising the amino
acid substitutions F42A, Y45A and L72G (numbering relative to the
human IL-2 sequence SEQ ID NO: 12).
21. The Type II anti-CD20 antibody of any one of the preceding
claims, wherein the therapeutic agent comprises an immunoconjugate
comprising an antibody as defined in claim 14, and a cytokine as
defined in claim 20.
22. The Type II anti-CD20 antibody of any one of claims 1 to 16,
wherein the therapeutic agent comprises a bispecific antibody
comprising an antibody as defined in claim 14 and an antibody as
defined in claim 16.
23. The Type II anti-CD20 antibody of any one of claims 1 to 12 and
16 to 18, wherein the therapeutic agent comprises a bispecific
antibody comprising an antibody as defined in claim 16 and an
antibody as defined in claim 20.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. patent application
Ser. No. 17/240,821, filed Apr. 26, 2021, which is a divisional of
U.S. patent application Ser. No. 15/371,891, filed Dec. 7, 2016,
now U.S. Pat. No. 11,013,801, which claims priority to European
Patent Application No. EP 16193151.4, filed Oct. 10, 2016, European
Patent Application No. EP 16172739.1, filed Jun. 2, 2016, and
European Patent Application No. EP 15198715.3, filed Dec. 9, 2015,
the disclosures of which are incorporated herein by reference in
their entirety.
SEQUENCE LISTING
[0002] The present application contains a Sequence Listing which
has been submitted in ASCII format via EFS-Web and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Nov. 4, 2021 is named 51177-018003_Sequence_Listing_11_4_21_ST25
and is 155,309 bytes in size.
FIELD OF THE INVENTION
[0003] The present invention relates to methods of treating a
disease, and methods for reduction of the formation of anti-drug
antibodies (ADAs) in response to the administration of a
therapeutic agent. The invention further relates to methods of
treating a disease, particularly a B-cell proliferative disorder,
and methods for reduction of adverse effects in response to the
administration of a therapeutic agent, particularly a T-cell
activating therapeutic agent.
BACKGROUND
[0004] The number of biotechnology-derived therapeutic agents
available for use in clinical settings has dramatically increased
in recent years, and includes recombinant human cytokines (e.g.
.alpha. and .beta. interferon, interleukin-2), cellular growth
factors (e.g. GM-CSF), hormones (e.g. glucagon), neuromuscular
antagonists (e.g. botulinum toxin), blood products (e.g. clotting
factor VIII), recombinant receptors (e.g. etanercept) and
monoclonal antibodies. Although therapeutic proteins are generally
considered safe and non-toxic, antibodies against these therapeutic
agents, known as anti-drug antibodies (ADAs), can develop during
treatment. ADAs have been observed in connection with various
therapeutic agents, such as erythropoietin, factor VIII, insulin,
immunotoxins and monoclonal antibodies (Schellekens and Casadevall,
J Neurol (2004), 251 [Suppl 2]:II/4-II/9; Mossoba et al., Clin
Cancer Res (2011) 17(11): 3697-3705; Hsu et al., British Journal of
Dermatology (2014) 170, 261-273). ADA formation is frequent for
example in autoimmune patients treated with TNF blockers and
impacts clinical outcome (Schaeverbecke et al., Rheumatology (2015)
doi: 10.1093/rheumatology/kev277).
[0005] The development of ADAs may influence serum concentrations
and function of therapeutic agents. The presence of ADAs may
increase clearance of the therapeutic agent through formation of
immune complexes between therapeutic agent and antibody
(neutralizing, non-neutralizing or both), thus reducing the
therapeutic agent's half-life. Furthermore, the activity and
effectiveness of the therapeutic agent may be decreased through
binding of antibody to the therapeutic agent. ADAs can also be
associated with allergic or hypersensitivity reactions and other
adverse events.
[0006] Since these adverse events associated with immune responses
can influence the safety and efficacy profile of therapeutics,
identification and development of strategies to overcome or inhibit
ADAs is of great interest.
[0007] Several protein engineering approaches have been
investigated to reduce the immunogenicity of protein therapeutics,
including for example masking or alteration of protein B cell
epitopes or modification of protein T cell epitopes. However,
clinical safety and success of these approaches has not been tested
and will require a significant degree of time to evaluate.
Therefore, there exists an immediate need to develop new
interventions using FDA-approved reagents to prevent ADA
responses.
[0008] Chemotherapy-based approaches aimed at host immune
suppression have been reported (Mossoba et al., Clin Cancer Res
(2011) 17(11): 3697-3705).
[0009] The anti-CD20 antibody rituximab has been used in
combination with methotrexate and intravenous immune globulin to
achieve tolerance to enzyme replacement therapy in a Morbus Pompe
patient (Mendelsohn et al., NEJM (2009) 360:2, 194-195). However,
in a clinical trial, host pretreatment with rituximab did not
inhibit the human immune response against the immunotoxin LMB-1
(Hassan et al., Clin Cancer Res (2004) 10, 16-18).
[0010] B-cell proliferative disorders describe a heterogeneous
group of malignancies that includes both leukemias and lymphomas.
Lymphomas develop from lymphatic cells and include two main
categories: Hodgkin lymphomas (HL) and the non-Hodgkin lymphomas
(NHL). In the United States, lymphomas of B cell origin constitute
approximately 80-85% of all non-Hodgkin lymphoma cases, and there
is considerable heterogeneity within the B-cell subset, based upon
genotypic and phenotypic expression patterns in the B-cell of
origin. For example, B cell lymphoma subsets include the
slow-growing indolent and incurable diseases, such as Follicular
lymphoma (FL) or chronic lymphocytic leukemia (CLL), as well as the
more aggressive subtypes, mantle cell lymphoma (MCL) and diffuse
large B cell lymphoma (DLBCL).
[0011] Despite the availability of various agents for the treatment
of B-cell proliferative disorders, there is an ongoing need for
development of safe and effective therapies to prolong remission
and improve cure rates in patients.
[0012] A strategy currently being investigated is the engagement of
T cells against malignant B cells. In order to effectively engage T
cells against malignant B cells, two recent approaches have been
developed. These two approaches are: 1) the administration of T
cells engineered ex vivo to recognize tumour cells (also known as
chimeric antigen receptor-modified T cell therapy [CAR-T cells])
(Maude et al., N Engl J Med (2014) 371,1507-1517); and, 2) the
administration of agents that activate endogenous T cells, such as
bispecific antibodies (Oak and Bartlett, Expert Opin Investig Drugs
(2015) 24, 715-724).
[0013] An example of the first approach is reported in the study by
Maude et al., in which 30 adult and pediatric patients were treated
with autologous T cells transduced with a CD19-directed chimeric
antigen receptor lentiviral vector (CTL019 CAR-T cells). The result
was a sustained remission based upon a 6-month event-free survival
rate of 67% and an overall survival rate of 78%. However, all
patients had cytokine release syndrome (CRS) (associated with
tumour burden), with 27% of patients having severe CRS. Central
nervous system toxicities of unknown cause were also noted at high
frequencies.
[0014] In contrast, the second approach, which involves activating
endogenous T cells to recognize tumour targets, bypasses this
hurdle of scalability, and can also provide competitive efficacy,
safety data and potentially long term durations of response. In
different CD20.sup.+ hematologic malignancies, this approach is
best exemplified by blinatumomab, a CD19 CD3 targeting T cell
bispecific molecule (Bargou et al., Science (2008) 321, 974-977)
that was recently approved for patients with minimal residual
disease-positive acute lymphocytic leukemia (ALL). This compound,
which is composed of two single chain Fv fragments (the so called
BiTE.RTM. format), directs the lysis of CD19.sup.+ cells by
cytolytic T cells. The primary constraint of blinatumomab is its
short half-life (approximately 2 hours), which necessitates
continuous infusion via a pump over 4-8 weeks. Nonetheless, it has
potent efficacy in patients with both relapsed/refractory
Non-Hodgkin Lymphoma (r/r NHL) and ALL, with step-up dosing (SUD)
required to mitigate severe cytokine release syndrome and CNS
toxicities (Nagorsen and Baeuerle, Exp Cell Res (2011) 317,
1255-1260).
[0015] The CD20 CD3 targeting T cell bispecific molecule, CD20XCD3
bsAB, is another example of a next generation of B cell targeting
antibody. CD20XCD3 bsAB is a T cell bispecific (TCB) antibody
targeting CD20 expressed on B cells and CD3 epsilon chain (CD3e)
present on T cells.
[0016] The mechanism of action of CD20XCD3 bsAB comprises
simultaneous binding to CD20.sup.+ B cells and CD3.sup.+ T cells,
leading to T-cell activation and T-cell mediated killing of B
cells. In the presence of CD20.sup.+ B cells, whether circulating
or tissue resident, pharmacologically active doses will trigger
T-cell activation and associated cytokine release. CD20XCD3 bsAB
has shown enhanced potency in nonclinical models over competitive T
cell engaging agents and, having an IgG-based format, has a greatly
improved half-life over blinatumomab.
[0017] Cytokine release is the result of activation of T cells. In
a phase 1 study conducted by TeGenero (Suntharalingam et al., N
Engl J Med (2006) 355,1018-1028), all 6 healthy volunteers
experienced near fatal, severe cytokine release syndrome (CRS)
rapidly post-infusion of an inappropriately-dosed, T-cell
stimulating super-agonist anti-CD28 monoclonal antibody. More
recently, in the above-mentioned study by Maude et al. of
CD19-targeting, chimeric antigen receptor T cell (CAR-T cell)
treatment of patients with relapsed ALL, all 30 patients had
cytokine release, which was categorized as severe in 27% of the
patients. CRS is a common but severe complication of CAR-T cell
therapy (reviewed in Xu and Tang, Cancer Letters (2014) 343,
172-178).
[0018] Severe CRS and CNS toxicity have also been frequently
observed with the CD19-CD3 T cell bispecific agent, blinatumomab
(Klinger et al., Blood. 2012; 119(26):6226-6233). In patients
receiving blinatumomab in all clinical trials, neurological
toxicities have occurred in approximately 50% of patients, and the
types of toxicities observed are well-defined in the package
insert.
[0019] It is not well understood if or how CNS toxicity is related
to earlier cytokine release or T cell activation. Similar to
blinatumomab, CNS AEs (ranging from delirium to global
encephalopathy) were reported for 43% (13/30) of the patients with
r/r ALL treated with CD19-targeting CAR-T cells (Maude et al., N
Engl J Med (2014) 371,1507-1517; Ghorashian et al., Br J Haematol
(2015) 169, 463-478). Neurologic toxic effects typically occurred
after symptoms of CRS had peaked and started to resolve; however no
direct, unequivocal association with severe CRS was found. The
authors proposed that the mechanism of neurotoxicity could involve
direct CAR-T-cell-mediated toxicity or it could be
cytokine-mediated. In contrast, an association between severe CRS
and neurotoxicity (e.g., encephalopathy) has been suggested in
another study of CD19-targeting CAR-T cell therapy (Davila et al.,
Sci Transl Med (2014) 6, 224ra25) and speculated to be due to
general T cell activation, versus direct CAR-T-induced damage.
[0020] Cytokine release and/or CNS-related toxicities are
particularly pronounced in T cell bispecific antibodies that link
CD3.sup.+ cells to B cells, as compared to other T cell bispecific
antibodies that link CD3.sup.+ cells to tissue-restricted (i.e.,
non-circulating) target cells. There is thus a need for methods to
reduce or prevent such adverse effects of these promising agents
which have the potential to significantly contribute to the
treatment of patients with B-cell proliferative disorders such as
NHL and CLL.
SUMMARY OF THE INVENTION
[0021] The present invention is based on the surprising finding
that (i) the formation of ADAs in response to administration of an
immunogenic therapeutic agent to a subject can effectively and
sustainably be prevented, and (ii) the cytokine release associated
with administration of a therapeutic agent, particularly a T-cell
activating therapeutic agent such as CD20XCD3 bsAB, to a subject
can be significantly reduced, by pre-treatment of said subject with
a Type II anti-CD20 antibody, such as obinutuzumab.
[0022] Obinutuzumab is a humanized glyco-engineered type II
anti-CD20 mAb that binds with high-affinity to the CD20 antigen,
inducing antibody-dependent cellular cytotoxicity (ADCC) and
antibody-dependent cellular phagocytosis (ADCP), low
complement-dependent cytotoxicity (CDC) activity, and high direct
cell death induction. To date, the safety profile of obinutuzumab
(including cytokine release) has been assessed and managed in
hundreds of patients in ongoing obinutuzumab clinical trials.
[0023] Without wishing to be bound by theory, the use of
obinutuzumab (GAZYVA.RTM.) pre-treatment (GPT) should aid in the
rapid depletion of B cells, both in the peripheral blood and in
secondary lymphoid organs, such that the risk of highly relevant
adverse events (AEs) from strong systemic T cell activation by
(T-cell activating) therapeutic agents (e.g. CRS) is reduced, while
supporting exposure levels of therapeutic agents that are high
enough from the start of dosing to mediate tumour cell elimination.
In addition to supporting the safety profile of (T-cell activating)
therapeutic agents such as CD20XCD3 bsAB, GPT should also help
prevent the formation of anti-drug antibodies (ADAs) to therapeutic
molecules.
[0024] For patients, GPT should translate into better drug exposure
with an enhanced safety profile.
[0025] GPT should be more effective in accomplishing the above
goals compared to other methods used, such as step up dosing (SUD).
For example, a single dose of obinutuzumab should allow
relapsed/refractory patients to receive the full therapeutic dose
of T-cell activating therapeutic agent such as CD20XCD3 bsAB, once
determined, without a time delay from step up dosing. In contrast
thereto, it was recently reported that the blinatumomab dosing
regimen for patients with r/r DLBCL in an ongoing Phase 2 trial
incorporates a double step up approach (i.e.,
9.fwdarw.28.fwdarw.112 .mu.g/m.sup.2/day), thus, requiring 14 days
to reach the maximum dose of 112 .mu.g/m.sup.2/day (Viardot el at.,
Hematol Oncol (2015) 33, 242 (Abstract 285)). As shown in the
Examples, following pretreatment with obinutuzumab, administration
of CD20XCD3 bsAB to cynomolgus monkeys was tolerated up to a level
that was ten times higher than that tolerated without GPT.
Efficient peripheral blood B-cell depletion and anti-tumour
activity along with strongly reduced cytokine release in the
peripheral blood associated with the first CD20XCD3 bsAB injection
was observed upon GPT.
[0026] Accordingly, in a first aspect the present invention
provides a method for (i) reducing the formation of anti-drug
antibodies (ADAs) against a therapeutic agent in a subject and/or
(ii) reducing cytokine release associated with administration of a
therapeutic agent, particularly a T-cell activating therapeutic
agent, in a subject, comprising administration of a Type II
anti-CD20 antibody to the subject prior to administration of the
therapeutic agent. In one embodiment the period of time between the
administration of the Type II anti-CD20 antibody and administration
of the therapeutic agent is sufficient for reduction of the number
of B-cells in the subject in response to the administration of the
Type II anti-CD20 antibody.
[0027] In a further aspect, the invention provides a method of
treating a disease in a subject, the method comprising a treatment
regimen comprising
[0028] (i) administration to the subject of a Type II anti-CD20
antibody,
[0029] and consecutively after a period of time
[0030] (ii) administration to the subject of a therapeutic
agent,
[0031] wherein the period of time between the administration of the
Type II anti-CD20 antibody and the administration of the
therapeutic agent is sufficient for reduction of the number of
B-cells in the subject in response to the administration of the
Type II anti-CD20 antibody.
[0032] In one embodiment, the treatment regimen effectively reduces
the formation of anti-drug antibodies (ADAs) in the subject in
response to the administration of the therapeutic agent as compared
to a corresponding treatment regimen without the administration of
the Type II anti-CD20 antibody.
[0033] In another embodiment, the treatment regimen effectively
reduces cytokine release associated with the administration of the
therapeutic agent in the subject as compared to a corresponding
treatment regimen without the administration of the Type II
anti-CD20 antibody. In such embodiment, the therapeutic agent
preferably is a T cell activating therapeutic agent.
[0034] In a further aspect, the invention provides a Type II
anti-CD20 antibody for use in a method for (i) reducing the
formation of anti-drug antibodies (ADAs) against a therapeutic
agent in a subject and/or (ii) reducing cytokine release associated
with the administration a therapeutic agent, particularly a T-cell
activating therapeutic agent, in a subject, comprising
administration of the Type II anti-CD20 antibody to the subject
prior to administration of the therapeutic agent.
[0035] In one embodiment, the period of time between the
administration of the Type II anti-CD20 antibody and administration
of the therapeutic agent is sufficient for reduction of the number
of B-cells in the subject in response to the administration of the
CD20 antibody.
[0036] In a further aspect, the invention provides a Type II
anti-CD20 antibody for use in a method of treating a disease in a
subject, the method comprising a treatment regimen comprising
[0037] (i) administration to the subject of the Type II anti-CD20
antibody,
[0038] and consecutively after a period of time
[0039] (ii) administration to the subject of a therapeutic
agent,
[0040] wherein the period of time between the administration of the
Type II anti-CD20 antibody and the administration of the
therapeutic agent is sufficient for reduction of the number of
B-cells in the subject in response to the administration of the
Type II anti-CD20 antibody.
[0041] In one embodiment, the treatment regimen effectively reduces
the formation of anti-drug antibodies (ADAs) against the
therapeutic agent in the subject (in response to the administration
of the therapeutic agent) as compared to a corresponding treatment
regimen without the administration of the anti-CD20 antibody.
[0042] In another embodiment, the treatment regimen effectively
reduces cytokine release associated with the administration of the
therapeutic agent in the subject as compared to a corresponding
treatment regimen without the administration of the Type II
anti-CD20 antibody. In such embodiment, the therapeutic agent
preferably is a T cell activating therapeutic agent.
[0043] In a further aspect, the invention provides the use of a
Type II anti-CD20 antibody in the manufacture of a medicament for
(i) reduction of the formation of anti-drug antibodies (ADAs)
against a therapeutic agent in a subject and/or (ii) the reduction
of cytokine release associated with administration of a therapeutic
agent, particularly a T-cell activating therapeutic agent, in a
subject,
[0044] wherein the medicament is to be used in a treatment regimen
comprising
[0045] (i) administration to the subject of the Type II anti-CD20
antibody,
[0046] and consecutively after a period of time
[0047] (ii) administration to the subject of a therapeutic
agent,
[0048] wherein the period of time between the administration of the
Type II anti-CD20 antibody and the administration of the
therapeutic agent is sufficient for reduction of the number of
B-cells in the subject in response to the administration of the
Type II anti-CD20 antibody.
[0049] In one embodiment, the treatment regimen effectively reduces
the formation of anti-drug antibodies (ADAs) against the
therapeutic agent in the subject as compared to a corresponding
treatment regimen without the administration of the anti-CD20
antibody.
[0050] In another embodiment, the treatment regimen effectively
reduces cytokine release associated with administration of the
therapeutic agent in the subject as compared to a corresponding
treatment regimen without the administration of the Type II
anti-CD20 antibody. In such embodiment, the therapeutic agent
preferably is a T cell activating therapeutic agent.
[0051] In still a further aspect, the invention provides a kit for
(i) the reduction of the formation of anti-drug antibodies (ADAs)
against a therapeutic agent in a subject and/or (ii) the reduction
of cytokine release associated with administration of a therapeutic
agent, particularly a T-cell activating therapeutic agent, in a
subject, comprising a package comprising a Type II anti-CD20
antibody composition and instructions for using the Type II
anti-CD20 antibody composition in a treatment regimen
comprising
[0052] (i) administration to the subject of the Type II anti-CD20
antibody composition,
[0053] and consecutively after a period of time
[0054] (ii) administration to the subject of a therapeutic
agent,
[0055] wherein the period of time between the administration of the
Type II anti-CD20 antibody composition and the administration of
the therapeutic agent is sufficient for reduction of the number of
B-cells in the subject in response to the administration of the
Type II CD20 antibody.
[0056] In one embodiment, the treatment regimen effectively reduces
the formation of anti-drug antibodies (ADAs) against the
therapeutic agent in the subject as compared to a corresponding
treatment regimen without the administration of the Type II
anti-CD20 antibody composition.
[0057] In another embodiment, the treatment regimen effectively
reduces cytokine release associated with administration of the
therapeutic agent in the subject as compared to a corresponding
treatment regimen without the administration of the Type II
anti-CD20 antibody composition. In such embodiment, the therapeutic
agent preferably is a T cell activating therapeutic agent. In one
embodiment, the kit further comprises a therapeutic agent
composition.
[0058] The invention in a further aspect as provides a therapeutic
agent for use in a method of treating a disease in a subject, the
method comprising a treatment regimen comprising
[0059] (i) administration to the subject of a Type II anti-CD20
antibody,
[0060] and consecutively after a period of time
[0061] (ii) administration to the subject of the therapeutic
agent,
[0062] wherein the period of time between the administration of the
Type II anti-CD20 antibody and the administration of the
therapeutic agent is sufficient for reduction of the number of
B-cells in the subject in response to the administration of the
CD20 antibody.
[0063] In one embodiment, the treatment regimen effectively reduces
the formation of anti-drug antibodies (ADAs) in the subject in
response to the administration of the therapeutic agent as compared
to a corresponding treatment regimen without the administration of
the Type II anti-CD20 antibody.
[0064] In another embodiment, the treatment regimen effectively
reduces cytokine release associated with administration of the
therapeutic agent in the subject as compared to a corresponding
treatment regimen without the administration of the Type II
anti-CD20 antibody. In such embodiment, the therapeutic agent
preferably is a T cell activating therapeutic agent.
[0065] The invention in still a further aspect provides the use of
a therapeutic agent in the manufacture of a medicament for
treatment of a disease in a subject, wherein the treatment
comprises a treatment regimen comprising
[0066] (i) administration to the subject of a Type II anti-CD20
antibody,
[0067] and consecutively after a period of time
[0068] (ii) administration to the subject of the therapeutic
agent,
[0069] wherein the period of time between the administration of the
Type II anti-CD20 antibody and the administration of the
therapeutic agent is sufficient for reduction of the number of
B-cells in the subject in response to the administration of the
Type II anti-CD20 antibody.
[0070] In one embodiment, the treatment regimen effectively reduces
the formation of anti-drug antibodies (ADAs) in the subject in
response to the administration of the therapeutic agent as compared
to a corresponding treatment regimen without the administration of
the Type II anti-CD20 antibody.
[0071] In another embodiment, the treatment regimen effectively
reduces cytokine release associated with administration of the
therapeutic agent in the subject as compared to a corresponding
treatment regimen without the administration of the Type II
anti-CD20 antibody. In such embodiment, the therapeutic agent
preferably is a T cell activating therapeutic agent.
[0072] The invention in a further aspect provides a kit for the
treatment of a disease in a subject, comprising a package
comprising a therapeutic agent composition and instructions for
using the therapeutic agent composition in a treatment regimen
comprising
[0073] (i) administration to the subject of a Type II anti-CD20
antibody,
[0074] and consecutively after a period of time
[0075] (ii) administration to the subject of the therapeutic agent
composition,
[0076] wherein the period of time between the administration of the
Type II anti-CD20 antibody and the administration of the
therapeutic agent composition is sufficient for reduction of the
number of B-cells in the subject in response to the administration
of the Type II anti-CD20 antibody.
[0077] In one embodiment, the treatment regimen effectively reduces
the formation of anti-drug antibodies (ADAs) against the
therapeutic agent in the subject as compared to a corresponding
treatment regimen without the administration of the Type II
anti-CD20 antibody composition.
[0078] In another embodiment, the treatment regimen effectively
reduces cytokine release associated with administration of the
therapeutic agent in the subject as compared to a corresponding
treatment regimen without the administration of the Type II
anti-CD20 antibody composition. In such embodiment, the therapeutic
agent preferably is a T cell activating therapeutic agent. In one
embodiment, the kit further comprises a Type II anti-CD20 antibody
composition.
[0079] The methods, uses, Type II anti-CD20 antibodies, therapeutic
agents and kits of the invention may incorporate, singly or in
combination, any of the features described hereinbelow.
[0080] In one embodiment, the Type II anti-CD20 antibody comprises
a heavy chain variable region comprising the heavy chain CDR (HCDR)
1 of SEQ ID NO: 4, the HCDR2 of SEQ ID NO: 5, and the HCDR3 of SEQ
ID NO: 6; and a light chain variable region comprising the light
chain CDR (LCDR) 1 of SEQ ID NO: 7, the LCDR2 of SEQ ID NO: 8 and
the LCDR3 of SEQ ID NO: 9.
[0081] In a more specific embodiment, the Type II anti-CD20
antibody comprises the heavy chain variable region sequence of SEQ
ID NO: 10 and the light chain variable region sequence of SEQ ID
NO: 11.
[0082] In one embodiment, the Type II anti-CD20 antibody is an IgG
antibody, particularly an IgG.sub.1 antibody.
[0083] In one embodiment, the Type II anti-CD20 antibody is
engineered to have an increased proportion of non-fucosylated
oligosaccharides in the Fc region as compared to a non-engineered
antibody. In one embodiment, at least about 40% of the N-linked
oligosaccharides in the Fc region of the Type II anti-CD20 antibody
are non-fucosylated.
[0084] In a particular embodiment the anti-CD20 antibody is
obinutuzumab.
[0085] In some embodiments, in particular in relation aspects of
the invention concerned with the reduction of the formation of
anti-drug antibodies (ADAs) against a therapeutic agent in a
subject, the therapeutic agent comprises a polypeptide.
[0086] In some embodiments, in particular in relation aspects of
the invention concerned with the reduction of the formation of
anti-drug antibodies (ADAs) against a therapeutic agent in a
subject, the therapeutic agent comprises an antibody.
[0087] In one such embodiment, the antibody specifically binds to
carcinoembryonic antigen (CEA). In one embodiment, the antibody
comprises a heavy chain variable region comprising the heavy chain
CDR (HCDR) 1 of SEQ ID NO: 14, the HCDR2 of SEQ ID NO: 15, and the
HCDR3 of SEQ ID NO: 16; and a light chain variable region
comprising the light chain CDR (LCDR) 1 of SEQ ID NO: 17, the LCDR2
of SEQ ID NO: 18 and the LCDR3 of SEQ ID NO: 19. In a further
embodiment, the antibody comprises the heavy chain variable region
sequence of SEQ ID NO: 20 and the light chain variable region
sequence of SEQ ID NO: 21. In another such embodiment, the antibody
specifically binds to CD3, particularly CD3 epsilon. In one
embodiment, the antibody comprises a heavy chain variable region
comprising the heavy chain CDR (HCDR) 1 of SEQ ID NO: 32, the HCDR2
of SEQ ID NO: 33, and the HCDR3 of SEQ ID NO: 34; and a light chain
variable region comprising the light chain CDR (LCDR) 1 of SEQ ID
NO: 35, the LCDR2 of SEQ ID NO: 36 and the LCDR3 of SEQ ID NO: 37.
In a further embodiment, the antibody comprises the heavy chain
variable region sequence of SEQ ID NO: 38 and the light chain
variable region sequence of SEQ ID NO: 39.
[0088] In some embodiments, in particular in relation aspects of
the invention concerned with the reduction of the formation of
anti-drug antibodies (ADAs) against a therapeutic agent in a
subject, the therapeutic agent comprises a cytokine.
[0089] In one such embodiment, the cytokine is interleukin-2
(IL-2).
[0090] In another such embodiment, the cytokine is a mutant human
IL-2 polypeptide comprising the amino acid substitutions F42A, Y45A
and L72G (numbering relative to the human IL-2 sequence SEQ ID NO:
12).
[0091] In some embodiments, in particular in relation aspects of
the invention concerned with the reduction of the formation of
anti-drug antibodies (ADAs) against a therapeutic agent in a
subject, the therapeutic agent comprises an immunoconjugate.
[0092] In one such embodiment, the immunoconjugate comprises (a) an
antibody that specifically binds to CEA and comprises a heavy chain
variable region comprising the heavy chain CDR (HCDR) 1 of SEQ ID
NO: 14, the HCDR2 of SEQ ID NO: 15, and the HCDR3 of SEQ ID NO: 16;
and a light chain variable region comprising the light chain CDR
(LCDR) 1 of SEQ ID NO: 17, the LCDR2 of SEQ ID NO: 18 and the LCDR3
of SEQ ID NO: 19, and (b) a mutant human IL-2 polypeptide
comprising the amino acid substitutions F42A, Y45A and L72G
(numbering relative to the human IL-2 sequence SEQ ID NO: 12).
[0093] In a particular such embodiment, the therapeutic agent
comprises cergutuzumab amunaleukin (CEA-IL2v).
[0094] In some embodiments, in particular in relation aspects of
the invention concerned with the reduction of the formation of
anti-drug antibodies (ADAs) against a therapeutic agent in a
subject, the therapeutic agent comprises a bispecific antibody that
specifically binds to CEA and to CD3.
[0095] In one such embodiment the therapeutic agent comprises a
bispecific antibody comprising
[0096] (i) an antigen binding moiety that specifically binds to CD3
and comprises a heavy chain variable region comprising the heavy
chain CDR (HCDR) 1 of SEQ ID NO: 32, the HCDR2 of SEQ ID NO: 33,
and the HCDR3 of SEQ ID NO: 34; and a light chain variable region
comprising the light chain CDR (LCDR) 1 of SEQ ID NO: 35, the LCDR2
of SEQ ID NO: 36 and the LCDR3 of SEQ ID NO: 37; and
[0097] (ii) an antigen binding moiety that specifically bind to CEA
and comprises a heavy chain variable region comprising the heavy
chain CDR (HCDR) 1 of SEQ ID NO: 14, the HCDR2 of SEQ ID NO: 15,
and the HCDR3 of SEQ ID NO: 16; and a light chain variable region
comprising the light chain CDR (LCDR) 1 of SEQ ID NO: 17, the LCDR2
of SEQ ID NO: 18 and the LCDR3 of SEQ ID NO: 19.
[0098] In a particular embodiment, the therapeutic agent comprises
CEA TCB.
[0099] In some embodiments, in particular in relation aspects of
the invention concerned with the reduction of cytokine release
associated with the administration of a therapeutic agent in a
subject, the therapeutic agent is a T cell activating therapeutic
agent.
[0100] In one embodiment, the T-cell activating therapeutic agent
comprises an antibody, particularly a multispecific (e.g. a
bispecific) antibody.
[0101] In one embodiment, the antibody specifically binds to an
activating T cell antigen.
[0102] In one embodiment, the antibody specifically binds to an
antigen selected from the group of CD3, CD28, CD137 (also known as
4-1BB), CD40, CD226, OX40, GITR, CD27 HVEM, and CD127.
[0103] In one embodiment, the antibody specifically binds to CD3,
particularly CD3.epsilon..
[0104] In one embodiment, the antibody comprises a heavy chain
variable region comprising the heavy chain CDR (HCDR) 1 of SEQ ID
NO: 32, the HCDR2 of SEQ ID NO: 33, and the HCDR3 of SEQ ID NO: 34;
and a light chain variable region comprising the light chain CDR
(LCDR) 1 of SEQ ID NO: 35, the LCDR2 of SEQ ID NO: 36 and the LCDR3
of SEQ ID NO: 37.
[0105] In one embodiment, the antibody comprises the heavy chain
variable region sequence of SEQ ID NO: 38 and the light chain
variable region sequence of SEQ ID NO: 39.
[0106] In one embodiment, the antibody specifically binds to a
B-cell antigen, particularly a malignant B-cell antigen.
[0107] In one embodiment, the antibody specifically binds to an
antigen selected from the group consisting of CD20, CD19, CD22,
ROR-1, CD37 and CD5, particularly to CD20 or CD19.
[0108] In one embodiment, the antibody specifically binds to
CD20.
[0109] In one embodiment, the antibody comprises a heavy chain
variable region comprising the heavy chain CDR (HCDR) 1 of SEQ ID
NO: 4, the HCDR2 of SEQ ID NO: 5, and the HCDR3 of SEQ ID NO: 6;
and a light chain variable region comprising the light chain CDR
(LCDR) 1 of SEQ ID NO: 7, the LCDR2 of SEQ ID NO: 8 and the LCDR3
of SEQ ID NO: 9.
[0110] In one embodiment, the antibody comprises the heavy chain
variable region sequence of SEQ ID NO: 10 and the light chain
variable region sequence of SEQ ID NO: 11.
[0111] In one embodiment, the antibody is a multispecific antibody,
particularly a bispecific antibody.
[0112] In one embodiment, the multispecific antibody specifically
binds to (i) an activating T cell antigen and (ii) a B cell
antigen.
[0113] In one embodiment, the multispecific antibody specifically
binds to (i) CD3 and (ii) an antigen selected from CD20 and
CD19.
[0114] In one embodiment, the multispecific antibody specifically
binds to CD3 and CD20.
[0115] In some embodiments, in particular in relation aspects of
the invention concerned with the reduction of cytokine release
associated with the administration of a therapeutic agent in a
subject, the therapeutic agent comprises a bispecific antibody
comprising
[0116] (i) an antigen binding moiety that specifically binds to CD3
and comprises a heavy chain variable region comprising the heavy
chain CDR (HCDR) 1 of SEQ ID NO: 32, the HCDR2 of SEQ ID NO: 33,
and the HCDR3 of SEQ ID NO: 34; and a light chain variable region
comprising the light chain CDR (LCDR) 1 of SEQ ID NO: 35, the LCDR2
of SEQ ID NO: 36 and the LCDR3 of SEQ ID NO: 37; and
[0117] (ii) an antigen binding moiety that specifically binds to
CD20 and comprises a heavy chain variable region comprising the
heavy chain CDR (HCDR) 1 of SEQ ID NO: 4, the HCDR2 of SEQ ID NO:
5, and the HCDR3 of SEQ ID NO: 6; and a light chain variable region
comprising the light chain CDR (LCDR) 1 of SEQ ID NO: 7, the LCDR2
of SEQ ID NO: 8 and the LCDR3 of SEQ ID NO: 9.
[0118] In a particular embodiment, the therapeutic agent comprises
CD20XCD3 bsAB.
[0119] In some embodiments, in particular in relation aspects of
the invention concerned with the reduction of cytokine release
associated with the administration of a therapeutic agent in a
subject, the therapeutic agent comprises a chimeric antigen
receptor (CAR) or a T cell expressing a CAR, particularly a CAR
that specifically binds to a B-cell antigen, more particularly a
CAR that specifically binds to an antigen selected from the group
of CD20, CD19, CD22, ROR-1, CD37 and CD5.
[0120] In some embodiments, in particular in relation aspects of
the invention concerned with the reduction of cytokine release
associated with the administration of a therapeutic agent in a
subject, the disease is a B cell proliferative disorder,
particularly a CD20-positive B-cell disorder. In one embodiment,
the disease is selected from the group consisting of Non-Hodgkin
lymphoma (NHL), acute lymphocytic leukemia (ALL), chronic
lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL),
follicular lymphoma (FL), mantle-cell lymphoma (MCL), marginal zone
lymphoma (MZL), Multiple myeloma (MM), and Hodgkin lymphoma
(HL).
BRIEF DESCRIPTION OF THE DRAWINGS
[0121] FIG. 1. Prior treatment with obinutuzumab but not rituximab
or vehicle results in the attenuation of tetanus toxoid specific de
novo IgG antibody responses in cynomolgus monkeys. Rituxan
indicates rituximab and GA101 obinutuzumab, respectively.
[0122] FIG. 2. Memory recall responses by measles specific IgG
antibody production in response to immune re-challenge with a
measles/rubella booster vaccination in animals with baseline titers
to measles is not affected by either obinutuzumab or rituximab in
cynomolgus monkeys. Rituxan indicates rituximab and GA101
obinutuzumab, respectively.
[0123] FIG. 3. B cell counts (CD45+CD19+) in peripheral blood
samples before start of obinutuzumab pre-treatment (BL=baseline),
before start of treatment with RO6895882 (C1D1=Cycle 1 Day 1) and
during treatment with RO6895882. Lines/symbols represent individual
patients. From the C1D1 time points onwards, no B cells were
detectable in the peripheral blood samples.
[0124] FIGS. 4A-C. Reduction of CD19+ cells (B cells) detected by
flow cytometry in tumor biopsies collected at baseline (BL) and
after treatment with obinutuzumab (treated). On-treatment samples
were obtained either before or during treatment with RO6895882. The
percentage of CD45+ cells (lymphocytes) staining positive for CD19
(B lymphocytes) was strongly reduced (B). No clear change was
observed for the percentage of CD16+ cells (Natural Killer Cells)
(A) or CD3+ cells (T lymphocytes) (C). Lines represent individual
patients.
[0125] FIGS. 5A-D. Reduction of B cells in tumor biopsies collected
at baseline (BL) and after treatment (treated) with obinutuzumab
measured by immunohistochemistry. On-treatment samples were
obtained either before or during treatment with RO6895882. The
density of B lymphocytes was measured by staining with CD20 (A, B)
and PA 5 (C, D). Both methods detected a depletion of B lymphocytes
in tumor and surrounding normal tissue. Lines represent individual
patients.
[0126] FIGS. 6A-Z. Exemplary configurations of the T cell
activating bispecific antigen binding molecules (TCBs) useful in
the invention. (A, D) Illustration of the "1+1 CrossMab" molecule.
(B, E) Illustration of the "2+1 IgG Crossfab" molecule with
alternative order of Crossfab and Fab components ("inverted"). (C,
F) Illustration of the "2+1 IgG Crossfab" molecule. (G, K)
Illustration of the "1+1 IgG Crossfab" molecule with alternative
order of Crossfab and Fab components ("inverted"). (H, L)
Illustration of the "1+1 IgG Crossfab" molecule. (I, M)
Illustration of the "2+1 IgG Crossfab" molecule with two CrossFabs.
(J, N) Illustration of the "2+1 IgG Crossfab" molecule with two
CrossFabs and alternative order of Crossfab and Fab components
("inverted"). (O, S) Illustration of the "Fab-Crossfab" molecule.
(P, T) Illustration of the "Crossfab-Fab" molecule. (Q, U)
Illustration of the "(Fab).sub.2-Crossfab" molecule. (R, V)
Illustration of the "Crossfab-(Fab).sub.2" molecule. (W, Y)
Illustration of the "Fab-(Crossfab).sub.2" molecule. (X, Z)
Illustration of the "(Crossfab).sub.2-Fab" molecule. Black dot:
optional modification in the Fc domain promoting
heterodimerization. ++, --: amino acids of opposite charges
optionally introduced in the CH1 and CL domains. Crossfab molecules
are depicted as comprising an exchange of VH and VL regions, but
may--in embodiments wherein no charge modifications are introduced
in CH1 and CL domains--alternatively comprise an exchange of the
CH1 and CL domains.
[0127] FIGS. 7A-B. B cell and T cell counts in the peripheral blood
in the different treatment groups. Flow cytometry analysis of
CD19.sup.+ B cells (A) and CD3.sup.+ T cells (B) in the peripheral
blood of vehicle and CD20XCD3 bsAB-treated fully humanized NOG
mice, 24 hours and 72 hours after first and second CD20XCD3 bsAB
administration. Black arrows indicate days of CD20XCD3 bsAB
administration.
[0128] FIGS. 8A-C. Cytokines released in peripheral blood among the
different treatment groups. Multiplex analysis of cytokines in
blood of vehicle and treated mice, 24 hours and 72 hours after the
first and second administration of CD20XCD3 bsAB. Histogram bars
represent the mean of 5 animals with error bars indicating the
standard deviation. Representative graphs for IFN.gamma. (A),
TNF.alpha. (B) and IL-6 (C) are shown. Compare the cytokine release
of the first injection of CD20XCD3 bsAB with and without
obinutuzumab pre-treatment (bars to be compared are indicated by
connecting lines).
[0129] FIG. 9. Anti-tumour activity of CD20XCD3 bsAB, obinutuzumab,
and obinutuzumab pretreatment (Gpt)+CD20XCD3 bsAB. Anti-tumour
activity of CD20XCD3 bsAB and obinutuzumab as monotherapy or
Gpt+CD20XCD3 bsAB in fully humanized NOG mice. Black arrow
indicates start of therapy. (8<n<10). Tumour model:
WSU-DLCL2.
[0130] FIGS. 10A-E. Cytokines released in peripheral blood of
cynomolgus monkeys following dosing with CD20XCD3 bsAB and
Gpt+CD20XCD3 bsAB treatments. (A) IFN.gamma., (B) IL-8, (C)
TNF.alpha., (D) IL-2, (E) IL-6.
[0131] FIG. 11. Anti-tumor activity upon step-up dosing of CD20XCD3
bsAB and obinutuzumab pretreatment (Gpt) in fully humanized NOG
mice bearing WSU-DLCL2 tumors. Mice received a first therapy
(arrow) either as a fractionated dose of CD20XCD3 bsAB (0.15, 0.05,
0.015 mg/kg IV) or Gpt (10 mg/kg obinutuzumab), followed by weekly
intravenous injections of CD20XCD3 bsAB at 0.5 mg/kg (full dose)
for 9 treatment cycles (i.e., 9 weeks). In the vehicle group, one
single mouse is shown from day 18. For the other groups, n=9 or 10.
Tumor model: WSU-DLCL2 injected subcutaneously. [CD20XCD3 bsAB 0.05
mg/kg+CD20XCD3 bsAB 0.05 mg/kg] vs [obinutuzumab 10 mg/kg+CD20XCD3
bsAB 0.5 mg/kg]*p=0.018 (One-way ANOVA analysis of sAUC with
Dunnet's method).
[0132] FIGS. 12A-H. T-cell staining in lungs from fully humanized
NOG mice bearing WSU-DLCL2 tumors, (A-D) 7 days after the first
treatment, and (E-H) 24 hours after the second treatment. Treatment
groups are as follows (single treatment or first+second treatment):
(A) vehicle, (B) obinutuzumab 10 mg/kg, (C) CD20XCD3 bsAB 0.15
mg/kg, (D) CD20XCD3 bsAB 0.5 mg/kg, (E) vehicle+vehicle, (F)
obinutuzumab 10 mg/kg+CD20XCD3 bsAB 0.5 mg/kg, (G) CD20XCD3 bsAB
0.15 mg/kg+CD20XCD3 bsAB 0.5 mg/kg, (H) vehicle+CD20XCD3 bsAB 0.5
mg/kg. Lung sections are immunohistochemically-stained with
anti-CD3 antibody (dark); nuclei were counterstained with
hematoxylin. Magnification 20.times.. Arrows point to increase in
perivascular CD3 positive cells.
[0133] FIGS. 13A-B. Lung of humanized NOG mouse sacrificed 24 hours
after single treatment with 0.5 mg/kg of CD20XCD3 bsAB. Margination
and adhesion of T cells (arrows) to the endothelium in vessels. Few
T cells have transmigrated to the perivascular space (asterisks).
(A) 20.times. magnification, (B) 40.times. magnification.
[0134] FIG. 14. Serum concentration-time curves of CD20XCD3 bsAB
following a single intravenous administration with or without
obinutuzumab Pretreatment in cynomolgus monkeys. Cynomolgus monkeys
were administered a single IV dose of 100-1000 .mu.g/kg CD20XCD3
bsAB with or without obinutuzumab pretreatment (Gpt) (50 mg/kg, 4
days prior to CD20XCD3 bsAB administration). Six animals are
represented in each dose group and the data is presented as
mean.+-.SD.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0135] Terms are used herein as generally used in the art, unless
otherwise defined in the following.
[0136] CD20 (also known as B-lymphocyte antigen CD20, B-lymphocyte
surface antigen B1, Leu-16, Bp35, BMS, and LF5; the human protein
is characterized in UniProt database entry P11836) is a hydrophobic
transmembrane protein with a molecular weight of approximately 35
kD expressed on pre-B and mature B lymphocytes (Valentine, M. A. et
al., J. Biol. Chem. 264 (1989) 11282-11287; Tedder, T. F., et al.,
Proc. Natl. Acad. Sci. U.S.A. 85 (1988) 208-212; Stamenkovic, I.,
et al., J. Exp. Med. 167 (1988) 1975-1980; Einfeld, D. A., et al.,
EMBO J. 7 (1988) 711-717; Tedder, T. F., et al., J. Immunol. 142
(1989) 2560-2568). The corresponding human gene is
Membrane-spanning 4-domains, subfamily A, member 1, also known as
MS4A1. This gene encodes a member of the membrane-spanning 4A gene
family. Members of this nascent protein family are characterized by
common structural features and similar intron/exon splice
boundaries and display unique expression patterns among
hematopoietic cells and nonlymphoid tissues. This gene encodes the
B-lymphocyte surface molecule which plays a role in the development
and differentiation of B-cells into plasma cells. This family
member is localized to 11q12, among a cluster of family members.
Alternative splicing of this gene results in two transcript
variants which encode the same protein.
[0137] The term "CD20" as used herein, refers to any native CD20
from any vertebrate source, including mammals such as primates
(e.g. humans) and rodents (e.g., mice and rats), unless otherwise
indicated. The term encompasses "full-length," unprocessed CD20 as
well as any form of CD20 that results from processing in the cell.
The term also encompasses naturally occurring variants of CD20,
e.g., splice variants or allelic variants. In one embodiment, CD20
is human CD20. The amino acid sequence of an exemplary human CD20
is shown in SEQ ID NO: 1.
[0138] The terms "anti-CD20 antibody" and "an antibody that binds
to CD20" refer to an antibody that is capable of binding CD20 with
sufficient affinity such that the antibody is useful as a
diagnostic and/or therapeutic agent in targeting CD20. In one
embodiment, the extent of binding of an anti-CD20 antibody to an
unrelated, non-CD20 protein is less than about 10% of the binding
of the antibody to CD20 as measured, e.g., by a radioimmunoassay
(RIA). In certain embodiments, an antibody that binds to CD20 has a
dissociation constant (Kd) of .ltoreq.1 .mu.M, .ltoreq.100 nM,
.ltoreq.10 nM, .ltoreq.1 nM, .ltoreq.0.1 nM, .ltoreq.0.01 nM, or
.ltoreq.0.001 nM (e.g. 10.sup.-8 M or less, e.g. from 10.sup.-8 M
to 10.sup.-13 M, e.g., from 10.sup.-9 M to 10.sup.-13 M). In
certain embodiments, an anti-CD20 antibody binds to an epitope of
CD20 that is conserved among CD20 from different species.
[0139] By "Type II anti-CD20 antibody" is meant an anti-CD20
antibody having binding properties and biological activities of
Type II anti-CD20 antibodies as described in Cragg et al., Blood
103 (2004) 2738-2743; Cragg et al., Blood 101 (2003) 1045-1052,
Klein et al., mAbs 5 (2013), 22-33, and summarized in Table 1
below.
TABLE-US-00001 TABLE 1 Properties of type I and type II anti-CD20
antibodies type I anti-CD20 antibodies type II anti-CD20 antibodies
Bind class I CD20 epitope Bind class II CD20 epitope Localize CD20
to lipid rafts Do not localize CD20 to lipid rafts High CDC * Low
CDC * ADCC activity * ADCC activity * Full binding capacity to B
cells Approx, half binding capacity to B cells Weak homotypic
aggregation Homotypic aggregation Low cell death induction Strong
cell death induction * if IgG.sub.1 isotype
[0140] Examples of type II anti-CD20 antibodies include e.g.
obinutuzumab (GA101), tositumumab (B1), humanized B-Ly1 antibody
IgG1 (a chimeric humanized IgG1 antibody as disclosed in WO
2005/044859), 11B8 IgG1 (as disclosed in WO 2004/035607) and AT80
IgG1.
[0141] Examples of type I anti-CD20 antibodies include e.g.
rituximab, ofatumumab, veltuzumab, ocaratuzumab, ocrelizumab,
PRO131921, ublituximab, HI47 IgG3 (ECACC, hybridoma), 2C6 IgG1 (as
disclosed in WO 2005/103081), 2F2 IgG1 (as disclosed in WO
2004/035607 and WO 2005/103081) and 2H7 IgG1 (as disclosed in WO
2004/056312).
[0142] The term "humanized B-Ly1 antibody" refers to humanized
B-Ly1 antibody as disclosed in WO 2005/044859 and WO 2007/031875,
which were obtained from the murine monoclonal anti-CD20 antibody
B-Ly1 (variable region of the murine heavy chain (VH): SEQ ID NO:
2; variable region of the murine light chain (VL): SEQ ID NO: 3
(see Poppema, S. and Visser, L., Biotest Bulletin 3 (1987) 131-139)
by chimerization with a human constant domain from IgG1 and
following humanization (see WO 2005/044859 and WO 2007/031875).
These "humanized B-Ly1 antibodies" are disclosed in detail in WO
2005/044859 and WO 2007/031875.
[0143] As used herein, the term "cytokine" refers to a molecule
that mediates and/or regulates a biological or cellular function or
process (e.g. immunity, inflammation, and hematopoiesis). The term
"cytokine" as used herein includes "lymphokines," "chemokines,"
"monokines," and "interleukins". Examples of useful cytokines
include, but are not limited to, GM-CSF, IL-1.alpha., IL-10, IL-2,
IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-15,
IFN-.alpha., IFN-.beta., IFN-.gamma., MIP-1.alpha., TGF-.beta.,
TNF-.alpha., and TNF-.beta.. A particular cytokines is IL-2. The
term "cytokine" as used herein is meant to also include cytokine
variants comprising one or more amino acid mutations in the amino
acid sequences of the corresponding wild-type cytokine, such as for
example the IL-2 variants described in Sauve et al., Proc Natl Acad
Sci USA 88, 4636-40 (1991); Hu et al., Blood 101, 4853-4861 (2003)
and US Pat. Publ. No. 2003/0124678; Shanafelt et al., Nature
Biotechnol 18, 1197-1202 (2000); Heaton et al., Cancer Res 53,
2597-602 (1993) and U.S. Pat. No. 5,229,109; US Pat. Publ. No.
2007/0036752; WO 2008/0034473; WO 2009/061853; or in WO
2012/107417.
[0144] The term "interleukin-2" or "IL-2" as used herein, refers to
any native IL-2 from any vertebrate source, including mammals such
as primates (e.g. humans) and rodents (e.g., mice and rats), unless
otherwise indicated. The term encompasses unprocessed IL-2 as well
as any form of IL-2 that results from processing in the cell. The
term also encompasses naturally occurring variants of IL-2, e.g.
splice variants or allelic variants. The amino acid sequence of an
exemplary human IL-2 is shown in SEQ ID NO: 12. Unprocessed human
IL-2 additionally comprises an N-terminal 20 amino acid signal
peptide having the sequence of SEQ ID NO: 31, which is absent in
the mature IL-2 molecule. The term "interleukin-2" as used herein
is meant to also include IL-2 variants comprising one or more amino
acid mutations in the amino acid sequences of the corresponding
wild-type cytokine, such as for example the IL-2 variants described
in Sauve et al., Proc Natl Acad Sci USA 88, 4636-40 (1991); Hu et
al., Blood 101, 4853-4861 (2003) and US Pat. Publ. No.
2003/0124678; Shanafelt et al., Nature Biotechnol 18, 1197-1202
(2000); Heaton et al., Cancer Res 53, 2597-602 (1993) and U.S. Pat.
No. 5,229,109; US Pat. Publ. No. 2007/0036752; WO 2008/0034473; WO
2009/061853; or in WO 2012/107417.
[0145] The term "IL-2 mutant" or "mutant IL-2 polypeptide" as used
herein is intended to encompass any mutant forms of various forms
of the IL-2 molecule including full-length IL-2, truncated forms of
IL-2 and forms where IL-2 is linked to another molecule such as by
fusion or chemical conjugation. "Full-length" when used in
reference to IL-2 is intended to mean the mature, natural length
IL-2 molecule. For example, full-length human IL-2 refers to a
molecule that has 133 amino acids (see e.g. SEQ ID NO: 12). The
various forms of IL-2 mutants are characterized in having a at
least one amino acid mutation affecting the interaction of IL-2
with CD25. This mutation may involve substitution, deletion,
truncation or modification of the wild-type amino acid residue
normally located at that position. Mutants obtained by amino acid
substitution are preferred. Unless otherwise indicated, an IL-2
mutant may be referred to herein as an IL-2 mutant peptide
sequence, an IL-2 mutant polypeptide, IL-2 mutant protein or IL-2
mutant analog. Designation of various forms of IL-2 is herein made
with respect to the sequence shown in SEQ ID NO: 12. Various
designations may be used herein to indicate the same mutation. For
example a mutation from phenylalanine at position 42 to alanine can
be indicated as 42A, A42, A42, F42A, or Phe42Ala.
[0146] As used herein, the term "release of cytokines" or "cytokine
release" is synonymous with "cytokine storm" or "cytokine release
syndrome" (abbreviated as "CRS"), and refers to an increase in the
levels of cytokines, particularly tumor necrosis factor alpha
(TNF-.alpha.), interferon gamma (IFN-.gamma.), interleukin-6
(IL-6), interleukin-10 (IL-10), interleukin-2 (IL-2) and/or
interleukin-8 (IL-8), in the blood of a subject during or shortly
after (e.g. within 1 day of) administration of a therapeutic agent,
resulting in adverse symptoms. Cytokine release is a type of
infusion-related reaction (IRR), which are common adverse drug
reactions to therapeutic agent and timely related to administration
of the therapeutic agent. IRRs typically occur during or shortly
after an administration of the therapeutic agent, i.e. typically
within 24 hours after infusion, predominantly at the first
infusion. In some instances, e.g. after the administration of CAR-T
cells, CRS can also occur only later, e.g. several days after
administration upon expansion of the CAR-T cells. The incidence and
severity typically decrease with subsequent infusions. Symptoms may
range from symptomatic discomfort to fatal events, and may include
fever, chills, dizziness, hypertension, hypotension, dyspnea,
restlessness, sweating, flushing, skin rash, tachycardia,
tachypnoea, headache, tumour pain, nausea, vomiting and/or organ
failure.
[0147] The term "amino acid mutation" as used herein is meant to
encompass amino acid substitutions, deletions, insertions, and
modifications. Any combination of substitution, deletion,
insertion, and modification can be made to arrive at the final
construct, provided that the final construct possesses the desired
characteristics, e.g., reduced binding to CD25 or to an Fc
receptor. Amino acid sequence deletions and insertions include
amino- and/or carboxy-terminal deletions and insertions of amino
acids. Particular amino acid mutations are amino acid
substitutions. For the purpose of altering e.g. the binding
characteristics of an IL-2 polypeptide or an Fc region,
non-conservative amino acid substitutions, i.e. replacing one amino
acid with another amino acid having different structural and/or
chemical properties, are particularly preferred. Amino acid
substitutions include replacement by non-naturally occurring amino
acids or by naturally occurring amino acid derivatives of the
twenty standard amino acids (e.g. 4-hydroxyproline,
3-methylhistidine, ornithine, homoserine, 5-hydroxylysine). Amino
acid mutations can be generated using genetic or chemical methods
well known in the art. Genetic methods may include site-directed
mutagenesis, PCR, gene synthesis and the like. It is contemplated
that methods of altering the side chain group of an amino acid by
methods other than genetic engineering, such as chemical
modification, may also be useful. Various designations may be used
herein to indicate the same amino acid mutation. For example, a
substitution from proline at position 329 of the Fc region to
glycine can be indicated as 329G, G329, G.sub.329, P329G, or
Pro329Gly.
[0148] The term "CD25" or ".alpha.-subunit of the IL-2 receptor" as
used herein, refers to any native CD25 from any vertebrate source,
including mammals such as primates (e.g. humans) and rodents (e.g.,
mice and rats), unless otherwise indicated. The term encompasses
"full-length", unprocessed CD25 as well as any form of CD25 that
results from processing in the cell. The term also encompasses
naturally occurring variants of CD25, e.g. splice variants or
allelic variants. In certain embodiments CD25 is human CD25. The
amino acid sequence of human CD25 is shown in UniProt
(www.uniprot.org) accession no. P01589, or NCBI
(www.ncbi.nlm.nih.gov/) RefSeq NP_000408.
[0149] The term "high-affinity IL-2 receptor" as used herein refers
to the heterotrimeric form of the IL-2 receptor, consisting of the
receptor .gamma.-subunit (also known as common cytokine receptor
.gamma.-subunit, .gamma..sub.c, or CD132), the receptor
.beta.-subunit (also known as CD122 or p70) and the receptor
.alpha.-subunit (also known as CD25 or p55). The term
"intermediate-affinity IL-2 receptor" by contrast refers to the
IL-2 receptor including only the .gamma.-subunit and the
.beta.-subunit, without the .alpha.-subunit (for a review see e.g.
Olejniczak and Kasprzak, Med Sci Monit 14, RA179-189 (2008)).
[0150] "Affinity" refers to the strength of the sum total of
non-covalent interactions between a single binding site of a
molecule (e.g., a receptor) and its binding partner (e.g., a
ligand). Unless indicated otherwise, as used herein, "binding
affinity" refers to intrinsic binding affinity which reflects a 1:1
interaction between members of a binding pair (e.g., receptor and a
ligand). The affinity of a molecule X for its partner Y can
generally be represented by the dissociation constant (K.sub.D),
which is the ratio of dissociation and association rate constants
(k.sub.off and k.sub.on, respectively). Thus, equivalent affinities
may comprise different rate constants, as long as the ratio of the
rate constants remains the same. Affinity can be measured by well
established methods known in the art. A particular method for
measuring affinity is Surface Plasmon Resonance (SPR).
[0151] "Reduction" (and grammatical variations thereof such as
"reduce" or "reducing"), for example reduction of the number of B
cells or the formation of ADAs or cytokine release, refers to a
decrease in the respective quantity, as measured by appropriate
methods known in the art. For clarity the term includes also
reduction to zero (or below the detection limit of the analytical
method), i.e. complete abolishment or elimination. Conversely,
"increased" refers to an increase in the respective quantity.
[0152] By "regulatory T cell" or "T.sub.reg cell" is meant a
specialized type of CD4.sup.+ T cell that can suppress the
responses of other T cells. T.sub.reg cells are characterized by
expression of the .alpha.-subunit of the IL-2 receptor (CD25) and
the transcription factor forkhead box P3 (FOXP3) (Sakaguchi, Annu
Rev Immunol 22, 531-62 (2004)) and play a critical role in the
induction and maintenance of peripheral self-tolerance to antigens,
including those expressed by tumors. T.sub.reg cells require IL-2
for their function and development and induction of their
suppressive characteristics.
[0153] As used herein, the term "antigen binding moiety" refers to
a polypeptide molecule that specifically binds to an antigenic
determinant. In one embodiment, an antigen binding moiety is able
to direct the entity to which it is attached (e.g. a cytokine or a
second antigen binding moiety) to a target site, for example to a
specific type of tumor cell or tumor stroma bearing the antigenic
determinant. Antigen binding moieties include antibodies and
fragments thereof as further defined herein. Preferred antigen
binding moieties include an antigen binding domain of an antibody,
comprising an antibody heavy chain variable region and an antibody
light chain variable region. In certain embodiments, the antigen
binding moieties may include antibody constant regions as further
defined herein and known in the art. Useful heavy chain constant
regions include any of the five isotypes: .alpha., .delta.,
.epsilon., .gamma., or .mu.. Useful light chain constant regions
include any of the two isotypes: .kappa. and .lamda..
[0154] By "specifically binds" is meant that the binding is
selective for the antigen and can be discriminated from unwanted or
non-specific interactions. The ability of an antigen binding moiety
to bind to a specific antigenic determinant can be measured either
through an enzyme-linked immunosorbent assay (ELISA) or other
techniques familiar to one of skill in the art, e.g. surface
plasmon resonance technique (analyzed on a BIAcore instrument)
(Liljeblad et al., Glyco J 17, 323-329 (2000)), and traditional
binding assays (Heeley, Endocr Res 28, 217-229 (2002)).
[0155] As used herein, the term "antigenic determinant" is
synonymous with "antigen" and "epitope," and refers to a site (e.g.
a contiguous stretch of amino acids or a conformational
configuration made up of different regions of non-contiguous amino
acids) on a polypeptide macromolecule to which an antigen binding
moiety binds, forming an antigen binding moiety-antigen complex.
Useful antigenic determinants can be found, for example, on the
surfaces of tumor cells, on the surfaces of virus-infected cells,
on the surfaces of other diseased cells, free in blood serum,
and/or in the extracellular matrix (ECM).
[0156] As used herein, term "polypeptide" refers to a molecule
composed of monomers (amino acids) linearly linked by amide bonds
(also known as peptide bonds). The term "polypeptide" refers to any
chain of two or more amino acids, and does not refer to a specific
length of the product. Thus, peptides, dipeptides, tripeptides,
oligopeptides, "protein," "amino acid chain," or any other term
used to refer to a chain of two or more amino acids, are included
within the definition of "polypeptide," and the term "polypeptide"
may be used instead of, or interchangeably with any of these terms.
The term "polypeptide" is also intended to refer to the products of
post-expression modifications of the polypeptide, including without
limitation glycosylation, acetylation, phosphorylation, amidation,
derivatization by known protecting/blocking groups, proteolytic
cleavage, or modification by non-naturally occurring amino acids. A
polypeptide may be derived from a natural biological source or
produced by recombinant technology, but is not necessarily
translated from a designated nucleic acid sequence. It may be
generated in any manner, including by chemical synthesis. A
polypeptide of the invention may be of a size of about 3 or more, 5
or more, 10 or more, 20 or more, 25 or more, 50 or more, 75 or
more, 100 or more, 200 or more, 500 or more, 1,000 or more, or
2,000 or more amino acids. Polypeptides may have a defined
three-dimensional structure, although they do not necessarily have
such structure. Polypeptides with a defined three-dimensional
structure are referred to as folded, and polypeptides which do not
possess a defined three-dimensional structure, but rather can adopt
a large number of different conformations, and are referred to as
unfolded.
[0157] By an "isolated" polypeptide or a variant, or derivative
thereof is intended a polypeptide that is not in its natural
milieu. No particular level of purification is required. For
example, an isolated polypeptide can be removed from its native or
natural environment. Recombinantly produced polypeptides and
proteins expressed in host cells are considered isolated for the
purpose of the invention, as are native or recombinant polypeptides
which have been separated, fractionated, or partially or
substantially purified by any suitable technique.
[0158] "Percent (%) amino acid sequence identity" with respect to a
reference polypeptide sequence is defined as the percentage of
amino acid residues in a candidate sequence that are identical with
the amino acid residues in the reference polypeptide sequence,
after aligning the sequences and introducing gaps, if necessary, to
achieve the maximum percent sequence identity, and not considering
any conservative substitutions as part of the sequence identity.
Alignment for purposes of determining percent amino acid sequence
identity can be achieved in various ways that are within the skill
in the art, for instance, using publicly available computer
software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR)
software. Those skilled in the art can determine appropriate
parameters for aligning sequences, including any algorithms needed
to achieve maximal alignment over the full length of the sequences
being compared. For purposes herein, however, % amino acid sequence
identity values are generated using the sequence comparison
computer program ALIGN-2. The ALIGN-2 sequence comparison computer
program was authored by Genentech, Inc., and the source code has
been filed with user documentation in the U.S. Copyright Office,
Washington D.C., 20559, where it is registered under U.S. Copyright
Registration No. TXU510087. The ALIGN-2 program is publicly
available from Genentech, Inc., South San Francisco, Calif., or may
be compiled from the source code. The ALIGN-2 program should be
compiled for use on a UNIX operating system, including digital UNIX
V4.0D. All sequence comparison parameters are set by the ALIGN-2
program and do not vary. In situations where ALIGN-2 is employed
for amino acid sequence comparisons, the % amino acid sequence
identity of a given amino acid sequence A to, with, or against a
given amino acid sequence B (which can alternatively be phrased as
a given amino acid sequence A that has or comprises a certain %
amino acid sequence identity to, with, or against a given amino
acid sequence B) is calculated as follows:
100 times the fraction X/Y
where X is the number of amino acid residues scored as identical
matches by the sequence alignment program ALIGN-2 in that program's
alignment of A and B, and where Y is the total number of amino acid
residues in B. It will be appreciated that where the length of
amino acid sequence A is not equal to the length of amino acid
sequence B, the % amino acid sequence identity of A to B will not
equal the % amino acid sequence identity of B to A. Unless
specifically stated otherwise, all % amino acid sequence identity
values used herein are obtained as described in the immediately
preceding paragraph using the ALIGN-2 computer program.
[0159] As used herein, the term "effector moiety" refers to a
polypeptide, e.g., a protein or glycoprotein, that influences
cellular activity, for example, through signal transduction or
other cellular pathways. Accordingly, the effector moiety can be
associated with receptor-mediated signaling that transmits a signal
from outside the cell membrane to modulate a response in a cell
bearing one or more receptors for the effector moiety. In one
embodiment, an effector moiety can elicit a cytotoxic response in
cells bearing one or more receptors for the effector moiety. In
another embodiment, an effector moiety can elicit a proliferative
response in cells bearing one or more receptors for the effector
moiety. In another embodiment, an effector moiety can elicit
differentiation in cells bearing receptors for the effector moiety.
In another embodiment, an effector moiety can alter expression
(i.e. upregulate or downregulate) of an endogenous cellular protein
in cells bearing receptors for the effector moiety. Non-limiting
examples of effector moieties include cytokines, growth factors,
hormones, enzymes, substrates, and cofactors. An effector moiety
can be associated with an antigen binding moiety such as an
antibody in a variety of configurations to form an
immunoconjugate.
[0160] The term "cytotoxic agent" as used herein refers to a
substance that inhibits or prevents a cellular function and/or
causes cell death or destruction. Cytotoxic agents include, but are
not limited to, radioactive isotopes (e.g., At.sup.211, I.sup.131,
I.sup.125, Y.sup.90, Re.sup.186, Re.sup.188, Sm.sup.153,
Bi.sup.212, P.sup.32, Pb.sup.212 and radioactive isotopes of Lu);
chemotherapeutic agents or drugs (e.g., methotrexate, adriamicin,
vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin,
melphalan, mitomycin C, chlorambucil, daunorubicin or other
intercalating agents); growth inhibitory agents; enzymes and
fragments thereof such as nucleolytic enzymes; antibiotics; toxins
such as small molecule toxins or enzymatically active toxins of
bacterial, fungal, plant or animal origin, including fragments
and/or variants thereof; and the various antitumor or anticancer
agents disclosed below.
[0161] The term "antibody" herein is used in the broadest sense and
encompasses various antibody structures, including but not limited
to monoclonal antibodies, polyclonal antibodies, multispecific
antibodies (e.g. bispecific antibodies), and antibody fragments so
long as they exhibit the desired antigen binding activity.
[0162] The terms "full length antibody," "intact antibody," and
"whole antibody" are used herein interchangeably to refer to an
antibody having a structure substantially similar to a native
antibody structure or having heavy chains that contain an Fc region
as defined herein.
[0163] An "antibody fragment" refers to a molecule other than an
intact antibody that comprises a portion of an intact antibody that
binds the antigen to which the intact antibody binds. Examples of
antibody fragments include but are not limited to Fv, Fab, Fab',
Fab'-SH, F(ab').sub.2, diabodies, linear antibodies, single-chain
antibody molecules (e.g. scFv), and multispecific antibodies formed
from antibody fragments. The term "antibody fragment" as used
herein also encompasses single-domain antibodies.
[0164] The term "immunoglobulin molecule" refers to a protein
having the structure of a naturally occurring antibody. For
example, immunoglobulins of the IgG class are heterotetrameric
glycoproteins of about 150,000 daltons, composed of two light
chains and two heavy chains that are disulfide-bonded. From N- to
C-terminus, each heavy chain has a variable region (VH), also
called a variable heavy domain or a heavy chain variable domain,
followed by three constant domains (CH1, CH2, and CH3), also called
a heavy chain constant region. Similarly, from N- to C-terminus,
each light chain has a variable region (VL), also called a variable
light domain or a light chain variable domain, followed by a
constant light (CL) domain, also called a light chain constant
region. The heavy chain of an immunoglobulin may be assigned to one
of five classes, called .alpha. (IgA), .delta. (IgD), .epsilon.
(IgE), .gamma. (IgG), or .mu. (IgM), some of which may be further
divided into subclasses, e.g. .gamma..sub.1 (IgG.sub.1),
.gamma..sub.2 (IgG.sub.2), .gamma..sub.3 (IgG.sub.3), .gamma..sub.4
(IgG.sub.4), .sigma..sub.1 (IgA.sub.1) and .alpha..sub.2
(IgA.sub.2). The light chain of an immunoglobulin may be assigned
to one of two types, called kappa (.kappa.) and lambda (.lamda.),
based on the amino acid sequence of its constant domain. An
immunoglobulin essentially consists of two Fab molecules and an Fc
domain, linked via the immunoglobulin hinge region.
[0165] The term "antigen binding domain" refers to the part of an
antibody that comprises the area which specifically binds to and is
complementary to part or all of an antigen. An antigen binding
domain may be provided by, for example, one or more antibody
variable domains (also called antibody variable regions).
Preferably, an antigen binding domain comprises an antibody light
chain variable region (VL) and an antibody heavy chain variable
region (VH).
[0166] The term "variable region" or "variable domain" refers to
the domain of an antibody heavy or light chain that is involved in
binding the antibody to antigen. The variable domains of the heavy
chain and light chain (VH and VL, respectively) of a native
antibody generally have similar structures, with each domain
comprising four conserved framework regions (FRs) and three
hypervariable regions (HVRs). See, e.g., Kindt et al., Kuby
Immunology, 6.sup.th ed., W.H. Freeman and Co., page 91 (2007). A
single VH or VL domain may be sufficient to confer antigen binding
specificity.
[0167] A "human antibody" is one which possesses an amino acid
sequence which corresponds to that of an antibody produced by a
human or a human cell or derived from a non-human source that
utilizes human antibody repertoires or other human
antibody-encoding sequences. This definition of a human antibody
specifically excludes a humanized antibody comprising non-human
antigen-binding residues.
[0168] A "humanized" antibody refers to a chimeric antibody
comprising amino acid residues from non-human HVRs and amino acid
residues from human FRs. In certain embodiments, a humanized
antibody will comprise substantially all of at least one, and
typically two, variable domains, in which all or substantially all
of the HVRs (e.g., CDRs) correspond to those of a non-human
antibody, and all or substantially all of the FRs correspond to
those of a human antibody. A humanized antibody optionally may
comprise at least a portion of an antibody constant region derived
from a human antibody. A "humanized form" of an antibody, e.g., a
non-human antibody, refers to an antibody that has undergone
humanization.
[0169] The term "hypervariable region" or "HVR" as used herein
refers to each of the regions of an antibody variable domain which
are hypervariable in sequence ("complementarity determining
regions" or "CDRs") and/or form structurally defined loops
("hypervariable loops") and/or contain the antigen-contacting
residues ("antigen contacts"). Generally, antibodies comprise six
HVRs: three in the VH (H1, H2, H3), and three in the VL (L1, L2,
L3). Exemplary HVRs herein include: [0170] (a) hypervariable loops
occurring at amino acid residues 26-32 (L1), 50-52 (L2), 91-96
(L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J.
Mol. Biol. 196:901-917 (1987)); [0171] (b) CDRs occurring at amino
acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1),
50-65 (H2), and 95-102 (H3) (Kabat et al., Sequences of Proteins of
Immunological Interest, 5th Ed. Public Health Service, National
Institutes of Health, Bethesda, Md. (1991)); [0172] (c) antigen
contacts occurring at amino acid residues 27c-36 (L1), 46-55 (L2),
89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et
al. J. Mol. Biol. 262: 732-745 (1996)); and [0173] (d) combinations
of (a), (b), and/or (c), including HVR amino acid residues 46-56
(L2), 47-56 (L2), 48-56 (L2), 49-56 (L2), 26-35 (H1), 26-35b (H1),
49-65 (H2), 93-102 (H3), and 94-102 (H3).
[0174] Unless otherwise indicated, HVR residues and other residues
in the variable domain (e.g., FR residues) are numbered herein
according to Kabat et al., supra.
[0175] "Framework" or "FR" refers to variable domain residues other
than hypervariable region (HVR) residues. The FR of a variable
domain generally consists of four FR domains: FR1, FR2, FR3, and
FR4. Accordingly, the HVR and FR sequences generally appear in the
following sequence in VH (or VL):
FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.
[0176] The "class" of an antibody refers to the type of constant
domain or constant region possessed by its heavy chain. There are
five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and
several of these may be further divided into subclasses (isotypes),
e.g., IgG.sub.1, IgG.sub.2, IgG.sub.3, IgG.sub.4, IgA.sub.1, and
IgA.sub.2. The heavy chain constant domains that correspond to the
different classes of immunoglobulins are called .alpha., .delta.,
.epsilon., .gamma., and .mu., respectively.
[0177] The term "Fc domain" or "Fc region" herein is used to define
a C-terminal region of an immunoglobulin heavy chain that contains
at least a portion of the constant region. The term includes native
sequence Fc regions and variant Fc regions. Although the boundaries
of the Fc region of an IgG heavy chain might vary slightly, the
human IgG heavy chain Fc region is usually defined to extend from
Cys226, or from Pro230, to the carboxyl-terminus of the heavy
chain. However, antibodies produced by host cells may undergo
post-translational cleavage of one or more, particularly one or
two, amino acids from the C-terminus of the heavy chain. Therefore
an antibody produced by a host cell by expression of a specific
nucleic acid molecule encoding a full-length heavy chain may
include the full-length heavy chain, or it may include a cleaved
variant of the full-length heavy chain (also referred to herein as
a "cleaved variant heavy chain"). This may be the case where the
final two C-terminal amino acids of the heavy chain are glycine
(G446) and lysine (K447, numbering according to Kabat EU index).
Therefore, the C-terminal lysine (Lys447), or the C-terminal
glycine (Gly446) and lysine (K447), of the Fc region may or may not
be present. Unless otherwise specified herein, numbering of amino
acid residues in the Fc region or constant region is according to
the EU numbering system, also called the EU index, as described in
Kabat et al., Sequences of Proteins of Immunological Interest, 5th
Ed. Public Health Service, National Institutes of Health, Bethesda,
Md., 1991 (see also above). A "subunit" of an Fc domain as used
herein refers to one of the two polypeptides forming the dimeric Fc
domain, i.e. a polypeptide comprising C-terminal constant regions
of an immunoglobulin heavy chain, capable of stable
self-association. For example, a subunit of an IgG Fc domain
comprises an IgG CH2 and an IgG CH3 constant domain.
[0178] A "modification promoting heterodimerization" is a
manipulation of the peptide backbone or the post-translational
modifications of a polypeptide, e.g. an immunoglobulin heavy chain,
that reduces or prevents the association of the polypeptide with an
identical polypeptide to form a homodimer. A modification promoting
heterodimerization as used herein particularly includes separate
modifications made to each of two polypeptides desired to form a
dimer, wherein the modifications are complementary to each other so
as to promote association of the two polypeptides. For example, a
modification promoting heterodimerization may alter the structure
or charge of one or both of the polypeptides desired to form a
dimer so as to make their association sterically or
electrostatically favorable, respectively. Heterodimerization
occurs between two non-identical polypeptides, such as two
immunoglobulin heavy chains wherein further immunoconjugate
components fused to each of the heavy chains (e.g. IL-2
polypeptide) are not the same. In the immunoconjugates useful in
the present invention, the modification promoting
heterodimerization is in the heavy chain(s), specifically in the Fc
domain, of an immunoglobulin molecule. In some embodiments the
modification promoting heterodimerziation comprises an amino acid
mutation, specifically an amino acid substitution. In a particular
embodiment, the modification promoting heterodimerization comprises
a separate amino acid mutation, specifically an amino acid
substitution, in each of the two immunoglobulin heavy chains.
[0179] Similarly, a "modification promoting the association of the
first and the second subunit of the Fc domain" is a manipulation of
the peptide backbone or the post-translational modifications of an
Fc domain subunit that reduces or prevents the association of a
polypeptide comprising the Fc domain subunit with an identical
polypeptide to form a homodimer. A modification promoting
association as used herein particularly includes separate
modifications made to each of the two Fc domain subunits desired to
associate (i.e. the first and the second subunit of the Fc domain),
wherein the modifications are complementary to each other so as to
promote association of the two Fc domain subunits. For example, a
modification promoting association may alter the structure or
charge of one or both of the Fc domain subunits so as to make their
association sterically or electrostatically favorable,
respectively. Thus, (hetero)dimerization occurs between a
polypeptide comprising the first Fc domain subunit and a
polypeptide comprising the second Fc domain subunit, which might be
non-identical in the sense that further components fused to each of
the subunits (e.g. antigen binding moieties) are not the same. In
some embodiments the modification promoting association comprises
an amino acid mutation in the Fc domain, specifically an amino acid
substitution. In a particular embodiment, the modification
promoting association comprises a separate amino acid mutation,
specifically an amino acid substitution, in each of the two
subunits of the Fc domain.
[0180] An "activating Fc receptor" is an Fc receptor that following
engagement by an Fc region of an antibody elicits signaling events
that stimulate the receptor-bearing cell to perform effector
functions. Activating Fc receptors include Fc.gamma.RIIIa (CD16a),
Fc.gamma.RI (CD64), Fc.gamma.RIIa (CD32), and Fc.alpha.RI
(CD89).
[0181] The term "effector functions" when used in reference to
antibodies refer to those biological activities attributable to the
Fc region of an antibody, which vary with the antibody isotype.
Examples of antibody effector functions include: C1q binding and
complement dependent cytotoxicity (CDC), Fc receptor binding,
antibody-dependent cell-mediated cytotoxicity (ADCC),
antibody-dependent cellular phagocytosis (ADCP), cytokine
secretion, immune complex-mediated antigen uptake by antigen
presenting cells, down regulation of cell surface receptors (e.g. B
cell receptor), and B cell activation.
[0182] As used herein, the term "effector cells" refers to a
population of lymphocytes that display effector moiety receptors,
e.g. cytokine receptors, and/or Fc receptors on their surface
through which they bind an effector moiety, e.g. a cytokine, and/or
an Fc region of an antibody and contribute to the destruction of
target cells, e.g. tumor cells. Effector cells may for example
mediate cytotoxic or phagocytic effects. Effector cells include,
but are not limited to, effector T cells such as CD8.sup.+
cytotoxic T cells, CD4.sup.+ helper T cells, .gamma..delta. T
cells, NK cells, lymphokine-activated killer (LAK) cells and
macrophages/monocytes.
[0183] As used herein, the terms "engineer, engineered,
engineering," are considered to include any manipulation of the
peptide backbone or the post-translational modifications of a
naturally occurring or recombinant polypeptide or fragment thereof.
Engineering includes modifications of the amino acid sequence, of
the glycosylation pattern, or of the side chain group of individual
amino acids, as well as combinations of these approaches.
"Engineering", particularly with the prefix "glyco-", as well as
the term "glycosylation engineering" includes metabolic engineering
of the glycosylation machinery of a cell, including genetic
manipulations of the oligosaccharide synthesis pathways to achieve
altered glycosylation of glycoproteins expressed in cells.
Furthermore, glycosylation engineering includes the effects of
mutations and cell environment on glycosylation. In one embodiment,
the glycosylation engineering is an alteration in
glycosyltransferase activity. In a particular embodiment, the
engineering results in altered glucosaminyltransferase activity
and/or fucosyltransferase activity. Glycosylation engineering can
be used to obtain a "host cell having increased GnTIII activity"
(e.g. a host cell that has been manipulated to express increased
levels of one or more polypeptides having
.beta.(1,4)-N-acetylglucosaminyltransferase III (GnTIII) activity),
a "host cell having increased ManII activity" (e.g. a host cell
that has been manipulated to express increased levels of one or
more polypeptides having .alpha.-mannosidase II (ManII) activity),
or a "host cell having decreased .alpha.(1,6) fucosyltransferase
activity" (e.g. a host cell that has been manipulated to express
decreased levels of .alpha.(1,6) fucosyltransferase).
[0184] The terms "host cell," "host cell line," and "host cell
culture" are used interchangeably and refer to cells into which
exogenous nucleic acid has been introduced, including the progeny
of such cells. Host cells include "transformants" and "transformed
cells," which include the primary transformed cell and progeny
derived therefrom without regard to the number of passages. Progeny
may not be completely identical in nucleic acid content to a parent
cell, but may contain mutations. Mutant progeny that have the same
function or biological activity as screened or selected for in the
originally transformed cell are included herein. A host cell is any
type of cellular system that can be used to generate proteins used
for the present invention. In one embodiment, the host cell is
engineered to allow the production of an antibody with modified
oligosaccharides. In certain embodiments, the host cells have been
manipulated to express increased levels of one or more polypeptides
having .beta.(1,4)-N-acetylglucosaminyltransferase III (GnTIII)
activity. In certain embodiments the host cells have been further
manipulated to express increased levels of one or more polypeptides
having .alpha.-mannosidase II (ManII) activity. Host cells include
cultured cells, e.g. mammalian cultured cells, such as CHO cells,
BHK cells, NS0 cells, SP2/0 cells, YO myeloma cells, P3X63 mouse
myeloma cells, PER cells, PER.C6 cells or hybridoma cells, yeast
cells, insect cells, and plant cells, to name only a few, but also
cells comprised within a transgenic animal, transgenic plant or
cultured plant or animal tissue.
[0185] As used herein, the term "polypeptide having GnTIII
activity" refers to polypeptides that are able to catalyze the
addition of a N-acetylglucosamine (GlcNAc) residue in .beta.-1,4
linkage to the .beta.-linked mannoside of the trimannosyl core of
N-linked oligosaccharides. This includes fusion polypeptides
exhibiting enzymatic activity similar to, but not necessarily
identical to, an activity of
.beta.(1,4)-N-acetylglucosaminyltransferase III, also known as
.beta.-1,4-mannosyl-glycoprotein 4-b
eta-N-acetylglucosaminyl-transferase (EC 2.4.1.144), according to
the Nomenclature Committee of the International Union of
Biochemistry and Molecular Biology (NC-IUBMB), as measured in a
particular biological assay, with or without dose dependency. In
the case where dose dependency does exist, it need not be identical
to that of GnTIII, but rather substantially similar to the
dose-dependency in a given activity as compared to the GnTIII (i.e.
the candidate polypeptide will exhibit greater activity or not more
than about 25-fold less and, preferably, not more than about
ten-fold less activity, and most preferably, not more than about
three-fold less activity relative to the GnTIII). In certain
embodiments the polypeptide having GnTIII activity is a fusion
polypeptide comprising the catalytic domain of GnTIII and the Golgi
localization domain of a heterologous Golgi resident polypeptide.
Particularly, the Golgi localization domain is the localization
domain of mannosidase II or GnTI, most particularly the
localization domain of mannosidase II. Alternatively, the Golgi
localization domain is selected from the group consisting of: the
localization domain of mannosidase I, the localization domain of
GnTII, and the localization domain of .alpha.1,6 core
fucosyltransferase. Methods for generating such fusion polypeptides
and using them to produce antibodies with increased effector
functions are disclosed in WO2004/065540, U.S. Provisional Pat.
Appl. No. 60/495,142 and U.S. Pat. Appl. Publ. No. 2004/0241817,
the entire contents of which are expressly incorporated herein by
reference.
[0186] As used herein, the term "Golgi localization domain" refers
to the amino acid sequence of a Golgi resident polypeptide which is
responsible for anchoring the polypeptide to a location within the
Golgi complex. Generally, localization domains comprise amino
terminal "tails" of an enzyme.
[0187] As used herein, the term "polypeptide having ManII activity"
refers to polypeptides that are able to catalyze the hydrolysis of
the terminal 1,3- and 1,6-linked .alpha.-D-mannose residues in the
branched GlcNAcMan.sub.5GlcNAc.sub.2 mannose intermediate of
N-linked oligosaccharides. This includes polypeptides exhibiting
enzymatic activity similar to, but not necessarily identical to, an
activity of Golgi .alpha.-mannosidase II, also known as mannosyl
oligosaccharide 1,3-1,6-.alpha.-mannosidase II (EC 3.2.1.114),
according to the Nomenclature Committee of the International Union
of Biochemistry and Molecular Biology (NC-IUBMB).
[0188] Antibody-dependent cell-mediated cytotoxicity (ADCC) is an
immune mechanism leading to the lysis of antibody-coated target
cells by immune effector cells. The target cells are cells to which
antibodies or fragments thereof comprising an Fc region
specifically bind, generally via the protein part that is
N-terminal to the Fc region. As used herein, the term
"increased/reduced ADCC" is defined as either an increase/reduction
in the number of target cells that are lysed in a given time, at a
given concentration of antibody in the medium surrounding the
target cells, by the mechanism of ADCC defined above, and/or a
reduction/increase in the concentration of antibody, in the medium
surrounding the target cells, required to achieve the lysis of a
given number of target cells in a given time, by the mechanism of
ADCC. The increase/reduction in ADCC is relative to the ADCC
mediated by the same antibody produced by the same type of host
cells, using the same standard production, purification,
formulation and storage methods (which are known to those skilled
in the art), but that has not been engineered. For example the
increase in ADCC mediated by an antibody produced by host cells
engineered to have an altered pattern of glycosylation (e.g. to
express the glycosyltransferase, GnTIII, or other
glycosyltransferases) by the methods described herein, is relative
to the ADCC mediated by the same antibody produced by the same type
of non-engineered host cells.
[0189] By "antibody having increased/reduced antibody dependent
cell-mediated cytotoxicity (ADCC)" is meant an antibody having
increased/reducedADCC as determined by any suitable method known to
those of ordinary skill in the art. One accepted in vitro ADCC
assay is as follows: [0190] 1) the assay uses target cells that are
known to express the target antigen recognized by the
antigen-binding region of the antibody; [0191] 2) the assay uses
human peripheral blood mononuclear cells (PBMCs), isolated from
blood of a randomly chosen healthy donor, as effector cells; [0192]
3) the assay is carried out according to following protocol: [0193]
i) the PBMCs are isolated using standard density centrifugation
procedures and are suspended at 5.times.10.sup.6 cells/ml in RPMI
cell culture medium; [0194] ii) the target cells are grown by
standard tissue culture methods, harvested from the exponential
growth phase with a viability higher than 90%, washed in RPMI cell
culture medium, labeled with 100 micro-Curies of .sup.51Cr, washed
twice with cell culture medium, and resuspended in cell culture
medium at a density of 10.sup.5 cells/ml; [0195] iii) 100
microliters of the final target cell suspension above are
transferred to each well of a 96-well microtiter plate; [0196] iv)
the antibody is serially-diluted from 4000 ng/ml to 0.04 ng/ml in
cell culture medium and 50 microliters of the resulting antibody
solutions are added to the target cells in the 96-well microtiter
plate, testing in triplicate various antibody concentrations
covering the whole concentration range above; [0197] v) for the
maximum release (MR) controls, 3 additional wells in the plate
containing the labeled target cells, receive 50 microliters of a 2%
(V/V) aqueous solution of non-ionic detergent (Nonidet, Sigma, St.
Louis), instead of the antibody solution (point iv above); [0198]
vi) for the spontaneous release (SR) controls, 3 additional wells
in the plate containing the labeled target cells, receive 50
microliters of RPMI cell culture medium instead of the antibody
solution (point iv above); [0199] vii) the 96-well microtiter plate
is then centrifuged at 50.times.g for 1 minute and incubated for 1
hour at 4.degree. C.; [0200] viii) 50 microliters of the PBMC
suspension (point i above) are added to each well to yield an
effector:target cell ratio of 25:1 and the plates are placed in an
incubator under 5% CO.sub.2 atmosphere at 37.degree. C. for 4
hours; [0201] ix) the cell-free supernatant from each well is
harvested and the experimentally released radioactivity (ER) is
quantified using a gamma counter; [0202] x) the percentage of
specific lysis is calculated for each antibody concentration
according to the formula (ER-MR)/(MR-SR).times.100, where ER is the
average radioactivity quantified (see point ix above) for that
antibody concentration, MR is the average radioactivity quantified
(see point ix above) for the MR controls (see point v above), and
SR is the average radioactivity quantified (see point ix above) for
the SR controls (see point vi above); [0203] 4) "increased/reduced
ADCC" is defined as either an increase/reduction in the maximum
percentage of specific lysis observed within the antibody
concentration range tested above, and/or a reduction/increase in
the concentration of antibody required to achieve one half of the
maximum percentage of specific lysis observed within the antibody
concentration range tested above. The increase/reduction in ADCC is
relative to the ADCC, measured with the above assay, mediated by
the same antibody, produced by the same type of host cells, using
the same standard production, purification, formulation and storage
methods, which are known to those skilled in the art, but that has
not been engineered.
[0204] As used herein, the term "immunoconjugate" refers to a
polypeptide molecule that includes at least one effector moiety,
such as a cytokine, and an antigen binding moiety, such as an
antibody. In certain embodiments, the immunoconjugate comprises not
more than one effector moiety. Particular immunoconjugates useful
in the invention essentially consist of one effector moiety and an
antibody joined by one or more peptide linkers. Particular
immunoconjugates according to the invention are fusion proteins,
i.e. the components of the immunoconjugate are joined by peptide
bonds.
[0205] The term "monoclonal antibody" as used herein refers to an
antibody obtained from a population of substantially homogeneous
antibodies, i.e., the individual antibodies comprising the
population are identical and/or bind the same epitope, except for
possible variant antibodies, e.g., containing naturally occurring
mutations or arising during production of a monoclonal antibody
preparation, such variants generally being present in minor
amounts. In contrast to polyclonal antibody preparations, which
typically include different antibodies directed against different
determinants (epitopes), each monoclonal antibody of a monoclonal
antibody preparation is directed against a single determinant on an
antigen. Thus, the modifier "monoclonal" indicates the character of
the antibody as being obtained from a substantially homogeneous
population of antibodies, and is not to be construed as requiring
production of the antibody by any particular method. For example,
the monoclonal antibodies to be used in accordance with the present
invention may be made by a variety of techniques, including but not
limited to the hybridoma method, recombinant DNA methods,
phage-display methods, and methods utilizing transgenic animals
containing all or part of the human immunoglobulin loci, such
methods and other exemplary methods for making monoclonal
antibodies being described herein.
[0206] As used herein, the terms "first", "second", "third" etc.
with respect to antigen binding moieties etc., are used for
convenience of distinguishing when there is more than one of each
type of moiety. Use of these terms is not intended to confer a
specific order or orientation unless explicitly so stated.
[0207] The terms "multispecific" and "bispecific" mean that the
antigen binding molecule is able to specifically bind to at least
two distinct antigenic determinants. Typically, a bispecific
antigen binding molecule comprises two antigen binding sites, each
of which is specific for a different antigenic determinant. In
certain embodiments a bispecific antigen binding molecule is
capable of simultaneously binding two antigenic determinants,
particularly two antigenic determinants expressed on two distinct
cells.
[0208] The term "valent" as used herein denotes the presence of a
specified number of antigen binding sites in an antigen binding
molecule. As such, the term "monovalent binding to an antigen"
denotes the presence of one (and not more than one) antigen binding
site specific for the antigen in the antigen binding molecule.
[0209] An "antigen binding site" refers to the site, i.e. one or
more amino acid residues, of an antigen binding molecule which
provides interaction with the antigen. For example, the antigen
binding site of an antibody comprises amino acid residues from the
complementarity determining regions (CDRs). A native immunoglobulin
molecule typically has two antigen binding sites, a Fab molecule
typically has a single antigen binding site.
[0210] A "T cell activating therapeutic agent" as used herein
refers to a therapeutic agent capable of inducing T cell activation
in a subject, particularly a therapeutic agent designed for
inducing T-cell activation in a subject. Examples of T cell
activating therapeutic agents include bispecific antibodies that
specifically bind an activating T cell antigen, such as CD3, and a
target cell antigen, such as CD20 or CD19. Further examples include
chimeric antigen receptors (CARs) which comprise a T cell
activating domain and an antigen binding moiety that specifically
binds to a target cell antigen, such as CD20 or CD19.
[0211] An "activating T cell antigen" as used herein refers to an
antigenic determinant expressed by a T lymphocyte, particularly a
cytotoxic T lymphocyte, which is capable of inducing or enhancing T
cell activation upon interaction with an antigen binding molecule.
Specifically, interaction of an antigen binding molecule with an
activating T cell antigen may induce T cell activation by
triggering the signaling cascade of the T cell receptor complex. An
exemplary activating T cell antigen is CD3.
[0212] "T cell activation" as used herein refers to one or more
cellular response of a T lymphocyte, particularly a cytotoxic T
lymphocyte, selected from: proliferation, differentiation, cytokine
secretion, cytotoxic effector molecule release, cytotoxic activity,
and expression of activation markers. The T cell activating
bispecific antigen binding molecules and T cell activating
therapeutic agents used in the present invention are capable of
inducing T cell activation. Suitable assays to measure T cell
activation are known in the art described herein.
[0213] A "target cell antigen" as used herein refers to an
antigenic determinant presented on the surface of a target cell,
for example a cell in a tumor such as a cancer cell or a cell of
the tumor stroma.
[0214] A "B-cell antigen" as used herein refers to an antigenic
determinant presented on the surface of a B lymphocyte,
particularly a malignant B lymphocyte (in that case the antigen
also being referred to as "malignant B-cell antigen").
[0215] A "T-cell antigen" as used herein refers to an antigenic
determinant presented on the surface of a T lymphocyte,
particularly a cytotoxic T lymphocyte.
[0216] A "Fab molecule" refers to a protein consisting of the VH
and CH1 domain of the heavy chain (the "Fab heavy chain") and the
VL and CL domain of the light chain (the "Fab light chain") of an
immunoglobulin.
[0217] By "chimeric antigen receptor" or "CAR" is meant a
genetically engineered receptor protein comprising an antigen
binding moiety, e.g. a single-chain variable fragment (scFv) of a
targeting antibody, a transmembrane domain, an intracellular T-cell
activating signaling domain (e.g. the CD3 zeta chain of the T-cell
receptor) and optionally one or more intracellular co-stimulatory
domains (e.g. of CD28, CD27, CD137 (4-1BB), Ox40). CARs mediate
antigen recognition, T cell activation, and--in the case of
second-generation CARs--costimulation to augment T cell
functionality and persistence. For a review see e.g. Jackson et
al., Nat Rev Clin Oncol (2016) 13, 370-383.
[0218] By "B cell proliferative disorder" is meant a disease
wherein the number of B cells in a patient is increased as compared
to the number of B cells in a healthy subject, and particularly
wherein the increase in the number of B cells is the cause or
hallmark of the disease. A "CD20-positive B cell proliferative
disorder" is a B cell proliferative disorder wherein B-cells,
particularly malignant B-cells (in addition to normal B-cells),
express CD20. Exemplary B cell proliferation disorders include
Non-Hodgkin lymphoma (NHL), acute lymphocytic leukemia (ALL),
chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma
(DLBCL), follicular lymphoma (FL), mantle-cell lymphoma (MCL),
marginal zone lymphoma (MZL), as well as some types of Multiple
myeloma (MM) and Hodgkin lymphoma (HL).
[0219] By "fused" is meant that the components (e.g. a Fab molecule
and an Fc domain subunit) are linked by peptide bonds, either
directly or via one or more peptide linkers.
[0220] An "anti-drug antibody" or "ADA" refers to an antibody that
binds to a therapeutic agent and may influence serum concentrations
and function of the therapeutic agent in a subject. The presence of
ADAs may increase clearance of the therapeutic agent through
formation of immune complexes between therapeutic agent and
antibody (neutralizing, non-neutralizing or both), thus reducing
the therapeutic agent's half-life. Furthermore, the activity and
effectiveness of the therapeutic agent may be decreased through
binding of antibody to the therapeutic agent (particularly in the
case of neutralizing ADAs). ADAs can also be associated with
allergic or hypersensitivity reactions and other adverse
events.
[0221] An "effective amount" of an agent refers to the amount that
is necessary to result in a physiological change in the cell or
tissue to which it is administered.
[0222] A "therapeutically effective amount" of an agent, e.g. a
pharmaceutical composition, refers to an amount effective, at
dosages and for periods of time necessary, to achieve the desired
therapeutic or prophylactic result. A therapeutically effective
amount of an agent for example eliminates, decreases, delays,
minimizes or prevents adverse effects of a disease.
[0223] By "therapeutic agent" is meant an active ingredient, e.g.
of a pharmaceutical composition, that is administered to a subject
in an attempt to alter the natural course of a disease in the
subject being treated, and can be performed either for prophylaxis
or during the course of clinical pathology. An "immunotherapeutic
agent" refers to a therapeutic agent that is administered to a
subject in an attempt to restore or enhance the subject's immune
response, e.g. to a tumor.
[0224] An "individual" or "subject" is a mammal. Mammals include,
but are not limited to, domesticated animals (e.g. cows, sheep,
cats, dogs, and horses), primates (e.g. humans and non-human
primates such as monkeys), rabbits, and rodents (e.g. mice and
rats). Preferably, the individual or subject is a human.
[0225] The term "pharmaceutical composition" refers to a
preparation which is in such form as to permit the biological
activity of an active ingredient contained therein to be effective,
and which contains no additional components which are unacceptably
toxic to a subject to which the composition would be
administered.
[0226] A "pharmaceutically acceptable carrier" refers to an
ingredient in a pharmaceutical composition, other than an active
ingredient, which is nontoxic to a subject. A pharmaceutically
acceptable carrier includes, but is not limited to, a buffer,
excipient, stabilizer, or preservative.
[0227] As used herein, "treatment" (and grammatical variations
thereof such as "treat" or "treating") refers to clinical
intervention in an attempt to alter the natural course of a disease
in the individual being treated, and can be performed either for
prophylaxis or during the course of clinical pathology. Desirable
effects of treatment include, but are not limited to, preventing
occurrence or recurrence of disease, alleviation of symptoms,
diminishment of any direct or indirect pathological consequences of
the disease, preventing metastasis, decreasing the rate of disease
progression, amelioration or palliation of the disease state, and
remission or improved prognosis. In some embodiments, methods of
the invention are used to delay development of a disease or to slow
the progression of a disease.
[0228] The term "package insert" is used to refer to instructions
customarily included in commercial packages of therapeutic
products, that contain information about the indications, usage,
dosage, administration, combination therapy, contraindications
and/or warnings concerning the use of such therapeutic
products.
[0229] "CD3" refers to any native CD3 from any vertebrate source,
including mammals such as primates (e.g. humans), non-human
primates (e.g. cynomolgus monkeys) and rodents (e.g. mice and
rats), unless otherwise indicated. The term encompasses
"full-length," unprocessed CD3 as well as any form of CD3 that
results from processing in the cell. The term also encompasses
naturally occurring variants of CD3, e.g., splice variants or
allelic variants. In one embodiment, CD3 is human CD3, particularly
the epsilon subunit of human CD3 (CD3.epsilon.). The amino acid
sequence of human CD3.epsilon. is shown in UniProt
(www.uniprot.org) accession no. P07766 (version 144), or NCBI
(www.ncbi.nlm.nih.gov/) RefSeq NP_000724.1. See also SEQ ID NO:
115. The amino acid sequence of cynomolgus [Macaca fascicularis]
CD3.epsilon. is shown in NCBI GenBank no. BAB71849.1. See also SEQ
ID NO: 116.
[0230] "CD19" refers to B-lymphocyte antigen CD19, also known as
B-lymphocyte surface antigen B4 or T-cell surface antigen Leu-12
and includes any native CD19 from any vertebrate source, including
mammals such as primates (e.g. humans) and rodents (e.g., mice and
rats), unless otherwise indicated. The term encompasses
"full-length," unprocessed CD19 as well as any form of CD19 that
results from processing in the cell. The term also encompasses
naturally occurring variants of CD19, e.g., splice variants or
allelic variants. In one embodiment, CD19 is human CD19. The amino
acid sequence of an exemplary human CD19 is shown in UniProt
(www.uniprot.org) accession no. P15391 (version 174), or NCBI
(www.ncbi.nlm.nih.gov/) RefSeq NP_001770.5, and SEQ ID NO: 117.
[0231] "Carcinoembryonic antigen" or "CEA" (also known as
Carcinoembryonic antigen-related cell adhesion molecule 5
(CEACAM5)) refers to any native CEA from any vertebrate source,
including mammals such as primates (e.g. humans), non-human
primates (e.g. cynomolgus monkeys) and rodents (e.g. mice and
rats), unless otherwise indicated. The term encompasses
"full-length," unprocessed CEA as well as any form of CEA that
results from processing in the cell. The term also encompasses
naturally occurring variants of CEA, e.g., splice variants or
allelic variants. In one embodiment, CEA is human CEA. The amino
acid sequence of human CEA is shown in UniProt (www.uniprot.org)
accession no. P06731, or NCBI (www.ncbi.nlm.nih.gov/) RefSeq
NP_004354.2.
[0232] "Fibroblast activation protein" or "FAP" (also known as
seprase) refers to any native FAP from any vertebrate source,
including mammals such as primates (e.g. humans), non-human
primates (e.g. cynomolgus monkeys) and rodents (e.g. mice and
rats), unless otherwise indicated. The term encompasses
"full-length," unprocessed FAP as well as any form of FAP that
results from processing in the cell. The term also encompasses
naturally occurring variants of FAP, e.g., splice variants or
allelic variants. In one embodiment, FAP is human FAP. The amino
acid sequence of human FAP is shown in UniProt (www.uniprot.org)
accession no. Q12884, or NCBI (www.ncbi.nlm.nih.gov/) RefSeq
NP_004451.2.
[0233] By a "crossover" Fab molecule (also termed "Crossfab") is
meant a Fab molecule wherein the variable domains or the constant
domains of the Fab heavy and light chain are exchanged (i.e.
replaced by each other), i.e. the crossover Fab molecule comprises
a peptide chain composed of the light chain variable domain VL and
the heavy chain constant domain 1 CH1 (VL-CH1, in N- to C-terminal
direction), and a peptide chain composed of the heavy chain
variable domain VH and the light chain constant domain CL (VH-CL,
in N- to C-terminal direction). For clarity, in a crossover Fab
molecule wherein the variable domains of the Fab light chain and
the Fab heavy chain are exchanged, the peptide chain comprising the
heavy chain constant domain 1 CH1 is referred to herein as the
"heavy chain" of the (crossover) Fab molecule. Conversely, in a
crossover Fab molecule wherein the constant domains of the Fab
light chain and the Fab heavy chain are exchanged, the peptide
chain comprising the heavy chain variable domain VH is referred to
herein as the "heavy chain" of the (crossover) Fab molecule.
[0234] In contrast thereto, by a "conventional" Fab molecule is
meant a Fab molecule in its natural format, i.e. comprising a heavy
chain composed of the heavy chain variable and constant domains
(VH-CH1, in N- to C-terminal direction), and a light chain composed
of the light chain variable and constant domains (VL-CL, in N- to
C-terminal direction).
[0235] Type II Anti-CD20 Antibodies
[0236] The CD20 molecule (also called human B-lymphocyte-restricted
differentiation antigen or Bp35) is a hydrophobic transmembrane
protein expressed on the surface of malignant and non-malignant
pre-B and mature B lymphocytes that has been described extensively
(Valentine, M. A., et al., J. Biol. Chem. 264 (1989) 11282-11287;
and Einfeld, D. A., et al., EMBO J. 7 (1988) 711-717; Tedder, T.
F., et al., Proc. Natl. Acad. Sci. U.S.A. 85 (1988) 208-212;
Stamenkovic, I., et al., J. Exp. Med. 167 (1988) 1975-1980; Tedder,
T. F., et al., J. Immunol. 142 (1989) 2560-2568).
[0237] CD20 is highly expressed by over 90% of B cell non-Hodgkin's
lymphomas (NHL) (Anderson, K. C., et al., Blood 63 (1984)
1424-1433) but is not found on hematopoietic stem cells, pro-B
cells, normal plasma cells, or other normal tissues (Tedder, T. F.,
et al., J, Immunol. 135 (1985) 973-979).
[0238] There exist two different types of anti-CD20 antibodies
differing significantly in their mode of CD20 binding and
biological activities (Cragg, M. S., et al., Blood 103 (2004)
2738-2743; and Cragg, M. S., et al., Blood 101 (2003) 1045-1052).
Type I anti-CD20 antibodies primarily utilize complement to kill
target cells, while Type II antibodies primarily operate through
direct induction of cell death.
[0239] Type I and Type II anti-CD20 antibodies and their
characteristics are reviewed e.g. in Klein et al., mAbs 5 (2013),
22-33. Type II anti-CD20 antibodies do not localize CD20 to lipid
rafts, show low CDC activity, show only about half the binding
capacity to B cells as compared to Type I anti-CD20 antibodies, and
induce homotypic aggregation and direct cell death. In contrast
thereto, Type I antibodies localize CD20 to lipid rafts, show high
CDC activity, full binding capacity to B cells, and only weak
induction of homotypic aggregation and direct cell death.
[0240] Obinutuzumab and tositumumab (CAS number 192391-48) are
examples of Type II anti-CD20 antibodies, while rituximab,
ofatumumab, veltuzumab, ocaratuzumab, ocrelizumab, PRO131921 and
ublituximab are examples of Type I anti-CD20 antibodies.
[0241] According to the invention, the anti-CD20 antibody is a Type
II anti-CD20 antibody. In one embodiment according to the present
invention, the Type II anti-CD20 antibody is capable of reducing
the number of B cells in a subject. In one embodiment the Type II
anti-CD20 antibody is an IgG antibody, particularly an IgG1
antibody. In one embodiment, the Type II anti-CD20 antibody is a
full-length antibody. In one embodiment, the Type II anti-CD20
antibody comprises an Fc region, particularly an IgG Fc region or,
more particularly, an IgG1 Fc region. In one embodiment the Type II
anti-CD20 antibody is a humanized B-Ly1 antibody. Particularly, the
Type II anti-CD20 antibody is a humanized, IgG-class Type II
anti-CD20 antibody, having the binding specificity of the murine
B-Ly1 antibody (Poppema and Visser, Biotest Bulletin 3, 131-139
(1987); SEQ ID NOs 2 and 3).
[0242] In one embodiment, the Type II anti-CD20 antibody comprises
a heavy chain variable region comprising the heavy chain CDR (HCDR)
1 of SEQ ID NO: 4, the HCDR2 of SEQ ID NO: 5, and the HCDR3 of SEQ
ID NO: 6; and a light chain variable region comprising the light
chain CDR (LCDR) 1 of SEQ ID NO: 7, the LCDR2 of SEQ ID NO: 8 and
the LCDR3 of SEQ ID NO: 9. Particularly, the heavy chain variable
region framework regions (FRs) FR1, FR2, and FR3 of said Type II
anti-CD20 antibody are human FR sequences encoded by the VH1_10
human germ-line sequence, the heavy chain variable region FR4 of
said anti-CD20 antibody is a human FR sequence encoded by the JH4
human germ-line sequence, the light chain variable region FRs FR1,
FR2, and FR3 of said Type II anti-CD20 antibody are human FR
sequences encoded by the VK_2_40 human germ-line sequence, and the
light chain variable region FR4 of said anti-CD20 antibody is a
human FR sequence encoded by the JK4 human germ-line sequence. In
one embodiment, the Type II anti-CD20 antibody comprises the heavy
chain variable region sequence of SEQ ID NO: 10 and the light chain
variable region sequence of SEQ ID NO: 11.
[0243] In a particular embodiment, the Type II anti-CD20 antibody
is obinutuzumab (recommended INN, WHO Drug Information, Vol. 26,
No. 4, 2012, p. 453). As used herein, obinutuzumab is synonymous
for GA101. The tradename is GAZYVA.RTM. or GAZYVARO.RTM.. This
replaces all previous versions (e.g. Vol. 25, No. 1, 2011,
p.'75-'76), and is formerly known as afutuzumab (recommended INN,
WHO Drug Information, Vol. 23, No. 2, 2009, p. 176; Vol. 22, No. 2,
2008, p. 124). In one embodiment, the Type II anti-CD20 antibody is
tositumomab.
[0244] The Type II anti-CD20 antibody useful in the present
invention may be engineered to have increased effector function, as
compared to a corresponding non-engineered antibody. In one
embodiment the antibody engineered to have increased effector
function has at least 2-fold, at least 10-fold or even at least
100-fold increased effector function, compared to a corresponding
non-engineered antibody. The increased effector function can
include, but is not limited to, one or more of the following:
increased Fc receptor binding, increased C1q binding and complement
dependent cytotoxicity (CDC), increased antibody-dependent
cell-mediated cytotoxicity (ADCC), increased antibody-dependent
cellular phagocytosis (ADCP), increased cytokine secretion,
increased immune complex-mediated antigen uptake by
antigen-presenting cells, increased binding to NK cells, increased
binding to macrophages, increased binding to monocytes, increased
binding to polymorphonuclear cells, increased direct signaling
inducing apoptosis, increased crosslinking of target-bound
antibodies, increased dendritic cell maturation, or increased T
cell priming.
[0245] In one embodiment the increased effector function one or
more selected from the group of increased Fc receptor binding,
increased CDC, increased ADCC, increased ADCP, and increased
cytokine secretion. In one embodiment the increased effector
function is increased binding to an activating Fc receptor. In one
such embodiment the binding affinity to the activating Fc receptor
is increased at least 2-fold, particularly at least 10-fold,
compared to the binding affinity of a corresponding non-engineered
antibody. In a specific embodiment the activating Fc receptor is
selected from the group of Fc.gamma.RIIIa, Fc.gamma.RI, and
Fc.gamma.RIIa. In one embodiment the activating Fc receptor is
Fc.gamma.RIIIa, particularly human Fc.gamma.RIIIa. In another
embodiment the increased effector function is increased ADCC. In
one such embodiment the ADCC is increased at least 10-fold,
particularly at least 100-fold, compared to the ADCC mediated by a
corresponding non-engineered antibody. In yet another embodiment
the increased effector function is increased binding to an
activating Fc receptor and increased ADCC.
[0246] Increased effector function can be measured by methods known
in the art. A suitable assay for measuring ADCC is described
herein. Other examples of in vitro assays to assess ADCC activity
of a molecule of interest are described in U.S. Pat. No. 5,500,362;
Hellstrom et al. Proc Natl Acad Sci USA 83, 7059-7063 (1986) and
Hellstrom et al., Proc Natl Acad Sci USA 82, 1499-1502 (1985); U.S.
Pat. No. 5,821,337; Bruggemann et al., J Exp Med 166, 1351-1361
(1987). Alternatively, non-radioactive assays methods may be
employed (see, for example, ACTI.TM. non-radioactive cytotoxicity
assay for flow cytometry (CellTechnology, Inc. Mountain View,
Calif.); and CytoTox 96.RTM. non-radioactive cytotoxicity assay
(Promega, Madison, Wis.)). Useful effector cells for such assays
include peripheral blood mononuclear cells (PBMC) and Natural
Killer (NK) cells. Alternatively, or additionally, ADCC activity of
the molecule of interest may be assessed in vivo, e.g. in a animal
model such as that disclosed in Clynes et al., Proc Natl Acad Sci
USA 95, 652-656 (1998). Binding to Fc receptors can be easily
determined e.g. by ELISA, or by Surface Plasmon Resonance (SPR)
using standard instrumentation such as a BIAcore instrument (GE
Healthcare), and Fc receptors such as may be obtained by
recombinant expression. According to a particular embodiment,
binding affinity to an activating Fc receptor is measured by
surface plasmon resonance using a BIACORE.RTM. T100 machine (GE
Healthcare) at 25.degree. C. Alternatively, binding affinity of
antibodies for Fc receptors may be evaluated using cell lines known
to express particular Fc receptors, such as NK cells expressing
Fc.gamma.IIIa receptor. C1q binding assays may also be carried out
to determine whether the antibody is able to bind C1q and hence has
CDC activity. See e.g., C1q and C3c binding ELISA in WO 2006/029879
and WO 2005/100402. To assess complement activation, a CDC assay
may be performed (see, for example, Gazzano-Santoro et al., J
Immunol Methods 202, 163 (1996); Cragg et al., Blood 101, 1045-1052
(2003); and Cragg and Glennie, Blood 103, 2738-2743 (2004)).
[0247] Increased effector function may result e.g. from
glycoengineering of the Fc region or the introduction of amino acid
mutations in the Fc region of the antibody. In one embodiment the
anti-CD20 antibody is engineered by introduction of one or more
amino acid mutations in the Fc region. In a specific embodiment the
amino acid mutations are amino acid substitutions. In an even more
specific embodiment the amino acid substitutions are at positions
298, 333, and/or 334 of the Fc region (EU numbering of residues).
Further suitable amino acid mutations are described e.g. in Shields
et al., J Biol Chem 9(2), 6591-6604 (2001); U.S. Pat. No.
6,737,056; WO 2004/063351 and WO 2004/099249. Mutant Fc regions can
be prepared by amino acid deletion, substitution, insertion or
modification using genetic or chemical methods well known in the
art. Genetic methods may include site-specific mutagenesis of the
encoding DNA sequence, PCR, gene synthesis, and the like. The
correct nucleotide changes can be verified for example by
sequencing.
[0248] In another embodiment the Type II anti-CD20 antibody is
engineered by modification of the glycosylation in the Fc region.
In a specific embodiment the Type II anti-CD20 antibody is
engineered to have an increased proportion of non-fucosylated
oligosaccharides in the Fc region as compared to a non-engineered
antibody. An increased proportion of non-fucosylated
oligosaccharides in the Fc region of an antibody results in the
antibody having increased effector function, in particular
increased ADCC.
[0249] In a more specific embodiment, at least about 20%, about
25%, about 30%, about 35%, about 40%, about 45%, about 50%, about
55%, about 60%, about 65%, about 70%, about 75%, about 80%, about
85%, about 90%, about 95%, or about 100%, preferably at least about
40%, of the N-linked oligosaccharides in the Fc region of the Type
II anti-CD20 antibody are non-fucosylated. In one embodiment,
between about 40% and about 80% of the N-linked oligosaccharides in
the Fc region of the Type II anti-CD20 antibody are
non-fucosylated. In one embodiment, between about 40% and about 60%
of the N-linked oligosaccharides in the Fc region of the Type II
anti-CD20 antibody are non-fucosylated. The non-fucosylated
oligosaccharides may be of the hybrid or complex type.
[0250] In another specific embodiment the Type II anti-CD20
antibody is engineered to have an increased proportion of bisected
oligosaccharides in the Fc region as compared to a non-engineered
antibody. In a more specific embodiment, at least about 10%, about
15%, about 20%, about 25%, about 30%, about 35%, about 40%, about
45%, about 50%, about 55%, about 60%, about 65%, about 70%, about
75%, about 80%, about 85%, about 90%, about 95%, or about 100%,
preferably at least about 40%, of the N-linked oligosaccharides in
the Fc region of the Type II anti-CD20 antibody are bisected. In
one embodiment, between about 40% and about 80% of the N-linked
oligosaccharides in the Fc region of the anti-CD20 antibody are
bisected. In one embodiment, between about 40% and about 60% of the
N-linked oligosaccharides in the Fc region of the Type II anti-CD20
antibody are bisected. The bisected oligosaccharides may be of the
hybrid or complex type.
[0251] In yet another specific embodiment the anti-CD20 antibody is
engineered to have an increased proportion of bisected,
non-fucosylated oligosaccharides in the Fc region, as compared to a
non-engineered antibody. In a more specific embodiment, at least
about 10%, about 15%, about 20%, about 25%, about 30%, about 35%,
about 40%, about 45%, about 50%, about 55%, about 60%, about 65%,
about 70%, about 75%, about 80%, about 85%, about 90%, about 95%,
or about 100%, preferably at least about 15%, more preferably at
least about 25%, of the N-linked oligosaccharides in the Fc region
of the anti-CD20 antibody are bisected, non-fucosylated. The
bisected, non-fucosylated oligosaccharides may be of the hybrid or
complex type.
[0252] The oligosaccharide structures in the antibody Fc region can
be analysed by methods well known in the art, e.g. by MALDI TOF
mass spectrometry as described in Umana et al., Nat Biotechnol 17,
176-180 (1999) or Ferrara et al., Biotechn Bioeng 93, 851-861
(2006). The percentage of non-fucosylated oligosaccharides is the
amount of oligosaccharides lacking fucose residues, relative to all
oligosaccharides attached to Asn 297 (e. g. complex, hybrid and
high mannose structures) and identified in an N-glycosidase F
treated sample by MALDI TOF MS. Asn 297 refers to the asparagine
residue located at about position 297 in the Fc region (EU
numbering of Fc region residues); however, Asn297 may also be
located about .+-.3 amino acids upstream or downstream of position
297, i.e., between positions 294 and 300, due to minor sequence
variations in antibodies. The percentage of bisected, or bisected
non-fucosylated, oligosaccharides is determined analogously.
[0253] In one embodiment the Type II anti-CD20 antibody is
engineered to have modified glycosylation in the Fc region, as
compared to a non-engineered antibody, by producing the antibody in
a host cell having altered activity of one or more
glycosyltransferase. Glycosyltransferases include
.beta.(1,4)-N-acetylglucosaminyltransferase III (GnTIII),
.beta.(1,4)-galactosyltransferase (GalT),
.beta.(1,2)-N-acetylglucosaminyltransferase I (GnTI),
.beta.(1,2)-N-acetylglucosaminyltransferase II (GnTII) and
.alpha.(1,6)-fucosyltransferase. In a specific embodiment the Type
II anti-CD20 antibody is engineered to have an increased proportion
of non-fucosylated oligosaccharides in the Fc region, as compared
to a non-engineered antibody, by producing the antibody in a host
cell having increased .beta.(1,4)-N-acetylglucosaminyltransferase
III (GnTIII) activity. In an even more specific embodiment the host
cell additionally has increased .alpha.-mannosidase II (ManII)
activity. The glycoengineering methodology that can be used for
engineering antibodies useful for the present invention has been
described in greater detail in Umana et al., Nat Biotechnol 17,
176-180 (1999); Ferrara et al., Biotechn Bioeng 93, 851-861 (2006);
WO 99/54342 (U.S. Pat. No. 6,602,684; EP 1071700); WO 2004/065540
(U.S. Pat. Appl. Publ. No. 2004/0241817; EP 1587921), WO 03/011878
(U.S. Pat. Appl. Publ. No. 2003/0175884), the entire content of
each of which is incorporated herein by reference in its entirety.
Antibodies glycoengineered using this methodology are referred to
as GlycoMabs herein.
[0254] Generally, any type of cultured cell line, including the
cell lines discussed herein, can be used to generate cell lines for
the production of anti-TNC A2 antibodies with altered glycosylation
pattern. Particular cell lines include CHO cells, BHK cells, NS0
cells, SP2/0 cells, YO myeloma cells, P3X63 mouse myeloma cells,
PER cells, PER.C6 cells or hybridoma cells, and other mammalian
cells. In certain embodiments, the host cells have been manipulated
to express increased levels of one or more polypeptides having
.beta.(1,4)-N-acetylglucosaminyltransferase III (GnTIII) activity.
In certain embodiments the host cells have been further manipulated
to express increased levels of one or more polypeptides having
.alpha.-mannosidase II (ManII) activity. In a specific embodiment,
the polypeptide having GnTIII activity is a fusion polypeptide
comprising the catalytic domain of GnTIII and the Golgi
localization domain of a heterologous Golgi resident polypeptide.
Particularly, said Golgi localization domain is the Golgi
localization domain of mannosidase II. Methods for generating such
fusion polypeptides and using them to produce antibodies with
increased effector functions are disclosed in Ferrara et al.,
Biotechn Bioeng 93, 851-861 (2006) and WO2004/065540, the entire
contents of which are expressly incorporated herein by
reference.
[0255] The host cells which contain the coding sequence of an
antibody useful for the invention and/or the coding sequence of
polypeptides having glycosyltransferase activity, and which express
the biologically active gene products may be identified e.g. by
DNA-DNA or DNA-RNA hybridization; the presence or absence of
"marker" gene functions; assessing the level of transcription as
measured by the expression of the respective mRNA transcripts in
the host cell; or detection of the gene product as measured by
immunoassay or by its biological activity--methods which are well
known in the art. GnTIII or Man II activity can be detected e.g. by
employing a lectin which binds to biosynthetis products of GnTIII
or ManII, respectively. An example for such a lectin is the
E.sub.4-PHA lectin which binds preferentially to oligosaccharides
containing bisecting GlcNAc. Biosynthesis products (i.e. specific
oligosaccharide structures) of polypeptides having GnTIII or ManII
activity can also be detected by mass spectrometric analysis of
oligosaccharides released from glycoproteins produced by cells
expressing said polypeptides. Alternatively, a functional assay
which measures the increased effector function, e.g. increased Fc
receptor binding, mediated by antibodies produced by the cells
engineered with the polypeptide having GnTIII or ManII activity may
be used.
[0256] In another embodiment the anti-CD20 antibody is engineered
to have an increased proportion of non-fucosylated oligosaccharides
in the Fc region, as compared to a non-engineered antibody, by
producing the antibody in a host cell having decreased
.alpha.(1,6)-fucosyltransferase activity. A host cell having
decreased .alpha.(1,6)-fucosyltransferase activity may be a cell in
which the .alpha.(1,6)-fucosyltransferase gene has been disrupted
or otherwise deactivated, e.g. knocked out (see Yamane-Ohnuki et
al., Biotech Bioeng 87, 614 (2004); Kanda et al., Biotechnol
Bioeng, 94(4), 680-688 (2006); Niwa et al., J Immunol Methods 306,
151-160 (2006)).
[0257] Other examples of cell lines capable of producing
defucosylated antibodies include Lec13 CHO cells deficient in
protein fucosylation (Ripka et al., Arch Biochem Biophys 249,
533-545 (1986); US Pat. Appl. No. US 2003/0157108; and WO
2004/056312, especially at Example 11). The antibodies useful in
the present invention can alternatively be glycoengineered to have
reduced fucose residues in the Fc region according to the
techniques disclosed in EP 1 176 195 A1, WO 03/084570, WO 03/085119
and U.S. Pat. Appl. Pub. Nos. 2003/0115614, 2004/093621,
2004/110282, 2004/110704, 2004/132140, U.S. Pat. No. 6,946,292
(Kyowa), e.g. by reducing or abolishing the activity of a
GDP-fucose transporter protein in the host cells used for antibody
production.
[0258] Glycoengineered antibodies useful in the invention may also
be produced in expression systems that produce modified
glycoproteins, such as those taught in WO 03/056914 (GlycoFi, Inc.)
or in WO 2004/057002 and WO 2004/024927 (Greenovation).
[0259] Therapeutic Agents
[0260] The present invention is useful in connection with various
therapeutic agents, particularly with therapeutic agents that are
immunogenic in the subject (i.e. have the ability of inducing an
immune response in the subject) and/or that activate T-cells in the
subject. Such therapeutic agents include, for example, recombinant
proteins.
[0261] In one embodiment, the therapeutic agent induces the
formation of ADAs in a subject when administered to the subject in
a treatment regimen without the administration of a Type II
anti-CD20 antibody. In one embodiment, the therapeutic agent
induces cytokine release in a subject when administered to the
subject in a treatment regimen without the administration of a Type
II anti-CD20 antibody. In one embodiment, the therapeutic agent
induces formation of ADAs and cytokine release in a subject when
administered to the subject in a treatment regimen without the
administration of a Type II anti-CD20 antibody.
[0262] In one embodiment, the therapeutic agent is a biologic
agent. In one embodiment, the therapeutic agent comprises a
polypeptide, particularly a recombinant polypeptide. In one
embodiment, the therapeutic agent comprises a polypeptide that does
not naturally occur in the subject and/or is immunogenic in the
subject. In one embodiment, the therapeutic agent is to be
systemically administered. In one embodiment, the therapeutic agent
is to be administered by infusion, particularly intravenous
infusion.
[0263] In one embodiment, the therapeutic agent comprises an
antigen binding polypeptide. In one embodiment, the therapeutic
agent comprises a polypeptide selected from the group of an
antibody, an antibody fragment, an Fc domain, and an
immunoconjugate. In one embodiment, the therapeutic agent comprises
a polypeptide selected from the group of an antibody, an antibody
fragment, an antigen receptor or an antigen-binding fragment
thereof, and a receptor ligand or a receptor-binding fragment
thereof. In one embodiment, the therapeutic agent comprises an
antibody. In one embodiment, the antibody is a monoclonal antibody.
In one embodiment, the antibody is a polyclonal antibody. In one
embodiment the antibody is a human antibody. In one embodiment, the
antibody is humanized antibody. In one embodiment the antibody is a
chimeric antibody. In one embodiment the antibody is full-length
antibody. In one embodiment the antibody is an IgG-class antibody,
particularly an IgG1 subclass antibody. In one embodiment, the
antibody is a recombinant antibody.
[0264] In certain embodiments, the therapeutic agent comprises an
antibody fragment. Antibody fragments include, but are not limited
to, Fab, Fab', Fab'-SH, F(ab')2, Fv, and scFv fragments, and other
fragments described below. For a review of certain antibody
fragments, see Hudson et al. Nat. Med. 9:129-134 (2003). For a
review of scFv fragments, see, e.g., Pluckthun, in The Pharmacology
of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds.,
(Springer-Verlag, New York), pp. 269-315 (1994); see also WO
93/16185; and U.S. Pat. Nos. 5,571,894 and 5,587,458. For
discussion of Fab and F(ab').sub.2 fragments comprising salvage
receptor binding epitope residues and having increased in vivo
half-life, see U.S. Pat. No. 5,869,046. In one embodiment, the
antibody fragment is a Fab fragment or a scFv fragment.
[0265] Diabodies are antibody fragments with two antigen-binding
sites that may be bivalent or bispecific. See, for example, EP
404,097; WO 1993/01161; Hudson et al., Nat. Med. 9:129-134 (2003);
and Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448
(1993). Triabodies and tetrabodies are also described in Hudson et
al., Nat. Med. 9:129-134 (2003).
[0266] Single-domain antibodies are antibody fragments comprising
all or a portion of the heavy chain variable domain or all or a
portion of the light chain variable domain of an antibody. In
certain embodiments, a single-domain antibody is a human
single-domain antibody (Domantis, Inc., Waltham, Mass.; see, e.g.,
U.S. Pat. No. 6,248,516 B1).
[0267] Antibody fragments can be made by various techniques,
including but not limited to proteolytic digestion of an intact
antibody as well as production by recombinant host cells (e.g. E.
coli or phage), as described herein.
[0268] In certain embodiments, the therapeutic agent comprises a
chimeric antibody. Certain chimeric antibodies are described, e.g.,
in U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad.
Sci. USA, 81:6851-6855 (1984)). In one example, a chimeric antibody
comprises a non-human variable region (e.g., a variable region
derived from a mouse, rat, hamster, rabbit, or non-human primate,
such as a monkey) and a human constant region. In a further
example, a chimeric antibody is a "class switched" antibody in
which the class or subclass has been changed from that of the
parent antibody. Chimeric antibodies include antigen-binding
fragments thereof.
[0269] In certain embodiments, the therapeutic agent comprises a
humanized antibody. Typically, a non-human antibody is humanized to
reduce immunogenicity to humans, while retaining the specificity
and affinity of the parental non-human antibody. Generally, a
humanized antibody comprises one or more variable domains in which
HVRs, e.g., CDRs, (or portions thereof) are derived from a
non-human antibody, and FRs (or portions thereof) are derived from
human antibody sequences. A humanized antibody optionally will also
comprise at least a portion of a human constant region. In some
embodiments, some FR residues in a humanized antibody are
substituted with corresponding residues from a non-human antibody
(e.g., the antibody from which the HVR residues are derived), e.g.,
to restore or improve antibody specificity or affinity.
[0270] Humanized antibodies and methods of making them are
reviewed, e.g., in Almagro and Fransson, Front. Biosci.
13:1619-1633 (2008), and are further described, e.g., in Riechmann
et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat'l Acad.
Sci. USA 86:10029-10033 (1989); U.S. Pat. Nos. 5,821,337,
7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods
36:25-34 (2005) (describing specificity determining region (SDR)
grafting); Padlan, Mol. Immunol. 28:489-498 (1991) (describing
"resurfacing"); Dall'Acqua et al., Methods 36:43-60 (2005)
(describing "FR shuffling"); and Osbourn et al., Methods 36:61-68
(2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000)
(describing the "guided selection" approach to FR shuffling).
[0271] Human framework regions that may be used for humanization
include but are not limited to: framework regions selected using
the "best-fit" method (see, e.g., Sims et al. J. Immunol. 151:2296
(1993)); framework regions derived from the consensus sequence of
human antibodies of a particular subgroup of light or heavy chain
variable regions (see, e.g., Carter et al. Proc. Natl. Acad. Sci.
USA, 89:4285 (1992); and Presta et al. J. Immunol., 151:2623
(1993)); human mature (somatically mutated) framework regions or
human germline framework regions (see, e.g., Almagro and Fransson,
Front. Biosci. 13:1619-1633 (2008)); and framework regions derived
from screening FR libraries (see, e.g., Baca et al., J. Biol. Chem.
272:10678-10684 (1997) and Rosok et al., J. Biol. Chem.
271:22611-22618 (1996)).
[0272] In certain embodiments, the therapeutic agent comprises a
human antibody. Human antibodies can be produced using various
techniques known in the art. Human antibodies are described
generally in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5:
368-74 (2001) and Lonberg, Curr. Opin. Immunol. 20:450-459
(2008).
[0273] Human antibodies may be prepared by administering an
immunogen to a transgenic animal that has been modified to produce
intact human antibodies or intact antibodies with human variable
regions in response to antigenic challenge. Such animals typically
contain all or a portion of the human immunoglobulin loci, which
replace the endogenous immunoglobulin loci, or which are present
extrachromosomally or integrated randomly into the animal's
chromosomes. In such transgenic mice, the endogenous immunoglobulin
loci have generally been inactivated. For review of methods for
obtaining human antibodies from transgenic animals, see Lonberg,
Nat. Biotech. 23:1117-1125 (2005). See also, e.g., U.S. Pat. Nos.
6,075,181 and 6,150,584 describing XENOMOUSE' technology; U.S. Pat.
No. 5,770,429 describing HuMAB.RTM. technology; U.S. Pat. No.
7,041,870 describing K-M MOUSE.RTM. technology, and U.S. Patent
Application Publication No. US 2007/0061900, describing
VELOCIMOUSE.RTM. technology). Human variable regions from intact
antibodies generated by such animals may be further modified, e.g.,
by combining with a different human constant region.
[0274] Human antibodies can also be made by hybridoma-based
methods. Human myeloma and mouse-human heteromyeloma cell lines for
the production of human monoclonal antibodies have been described.
(See, e.g., Kozbor J. Immunol., 133: 3001 (1984); Brodeur et al.,
Monoclonal Antibody Production Techniques and Applications, pp.
51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J.
Immunol., 147: 86 (1991).) Human antibodies generated via human
B-cell hybridoma technology are also described in Li et al., Proc.
Natl. Acad. Sci. USA, 103:3557-3562 (2006). Additional methods
include those described, for example, in U.S. Pat. No. 7,189,826
(describing production of monoclonal human IgM antibodies from
hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268
(2006) (describing human-human hybridomas). Human hybridoma
technology (Trioma technology) is also described in Vollmers and
Brandlein, Histology and Histopathology, 20(3):927-937 (2005) and
Vollmers and Brandlein, Methods and Findings in Experimental and
Clinical Pharmacology, 27(3): 185-91 (2005).
[0275] Human antibodies may also be generated by isolating Fv clone
variable domain sequences selected from human-derived phage display
libraries. Such variable domain sequences may then be combined with
a desired human constant domain. Techniques for selecting human
antibodies from antibody libraries are described below.
[0276] Antibodies comprised in the therapeutic agent may be
isolated by screening combinatorial libraries for antibodies with
the desired activity or activities. For example, a variety of
methods are known in the art for generating phage display libraries
and screening such libraries for antibodies possessing the desired
binding characteristics. Such methods are reviewed, e.g., in
Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien
et al., ed., Human Press, Totowa, N.J., 2001) and further
described, e.g., in the McCafferty et al., Nature 348:552-554;
Clackson et al., Nature 352: 624-628 (1991); Marks et al., J Mol.
Biol. 222: 581-597 (1992); Marks and Bradbury, in Methods in
Molecular Biology 248:161-175 (Lo, ed., Human Press, Totowa, N.J.,
2003); Sidhu et al., J. Mol. Biol. 338(2): 299-310 (2004); Lee et
al., J Mol. Biol. 340(5): 1073-1093 (2004); Fellouse, Proc. Natl.
Acad. Sci. USA 101(34): 12467-12472 (2004); and Lee et al., J.
Immunol. Methods 284(1-2): 119-132(2004).
[0277] In certain phage display methods, repertoires of VH and VL
genes are separately cloned by polymerase chain reaction (PCR) and
recombined randomly in phage libraries, which can then be screened
for antigen-binding phage as described in Winter et al., Ann. Rev.
Immunol., 12: 433-455 (1994). Phage typically display antibody
fragments, either as single-chain Fv (scFv) fragments or as Fab
fragments. Libraries from immunized sources provide high-affinity
antibodies to the immunogen without the requirement of constructing
hybridomas. Alternatively, the naive repertoire can be cloned
(e.g., from human) to provide a single source of antibodies to a
wide range of non-self and also self-antigens without any
immunization as described by Griffiths et al., EMBO J, 12: 725-734
(1993). Finally, naive libraries can also be made synthetically by
cloning unrearranged V-gene segments from stem cells, and using PCR
primers containing random sequence to encode the highly variable
CDR3 regions and to accomplish rearrangement in vitro, as described
by Hoogenboom and Winter, J Mol. Biol., 227: 381-388 (1992). Patent
publications describing human antibody phage libraries include, for
example: U.S. Pat. No. 5,750,373, and US Patent Publication Nos.
2005/0079574, 2005/0119455, 2005/0266000, 2007/0117126,
2007/0160598, 2007/0237764, 2007/0292936, and 2009/0002360.
[0278] Antibodies or antibody fragments isolated from human
antibody libraries are considered human antibodies or human
antibody fragments herein.
[0279] In certain embodiments, the therapeutic agent comprises a
multispecific antibody, e.g. a bispecific antibody. Multispecific
antibodies are monoclonal antibodies that have binding
specificities for at least two different sites. In certain
embodiments, the binding specificities are for different antigens.
In certain embodiments, the binding specificities are for different
epitopes on the same antigen. Bispecific antibodies may also be
used to localize cytotoxic agents to cells which express an
antigen. Bispecific antibodies can be prepared as full length
antibodies or antibody fragments.
[0280] Techniques for making multispecific antibodies include, but
are not limited to, recombinant co-expression of two immunoglobulin
heavy chain-light chain pairs having different specificities (see
Milstein and Cuello, Nature 305: 537 (1983)), WO 93/08829, and
Traunecker et al., EMBO J. 10: 3655 (1991)), and "knob-in-hole"
engineering (see, e.g., U.S. Pat. No. 5,731,168). Multi-specific
antibodies may also be made by engineering electrostatic steering
effects for making antibody Fc-heterodimeric molecules (WO
2009/089004A1); cross-linking two or more antibodies or fragments
(see, e.g., U.S. Pat. No. 4,676,980, and Brennan et al., Science,
229: 81 (1985)); using leucine zippers to produce bi-specific
antibodies (see, e.g., Kostelny et al., J. Immunol.,
148(5):1547-1553 (1992)); using "diabody" technology for making
bispecific antibody fragments (see, e.g., Hollinger et al., Proc.
Natl. Acad. Sci. USA, 90:6444-6448 (1993)); and using single-chain
Fv (sFv) dimers (see, e.g. Gruber et al., J. Immunol., 152:5368
(1994)); and preparing trispecific antibodies as described, e.g.,
in Tutt et al. J. Immunol. 147: 60 (1991).
[0281] Engineered antibodies with three or more functional antigen
binding sites, including "Octopus antibodies," are also included
herein (see, e.g. US 2006/0025576A1).
[0282] The antibody or fragment herein also includes a "Dual Acting
FAb" or "DAF" comprising an antigen binding site that binds to two
different antigens (see, US 2008/0069820, for example).
[0283] "Crossmab" antibodies are also included herein (see e.g.
WO2009080251, WO2009080252, WO2009080253, WO2009080254).
[0284] Another technique for making bispecific antibody fragments
is the "bispecific T cell engager" or BITE.RTM. approach (see,
e.g., WO2004/106381, WO2005/061547, WO2007/042261, and
WO2008/119567). This approach utilizes two antibody variable
domains arranged on a single polypeptide. For example, a single
polypeptide chain includes two single chain Fv (scFv) fragments,
each having a variable heavy chain (VH) and a variable light chain
(VL) domain separated by a polypeptide linker of a length
sufficient to allow intramolecular association between the two
domains. This single polypeptide further includes a polypeptide
spacer sequence between the two scFv fragments. Each scFv
recognizes a different epitope, and these epitopes may be specific
for different cell types, such that cells of two different cell
types are brought into close proximity or tethered when each scFv
is engaged with its cognate epitope. One particular embodiment of
this approach includes a scFv recognizing a cell-surface antigen
expressed by an immune cell, e.g., a CD3 polypeptide on a T cell,
linked to another scFv that recognizes a cell-surface antigen
expressed by a target cell, such as a malignant or tumor cell.
[0285] As it is a single polypeptide, the bispecific T cell engager
may be expressed using any prokaryotic or eukaryotic cell
expression system known in the art, e.g., a CHO cell line. However,
specific purification techniques (see, e.g., EP1691833) may be
necessary to separate monomeric bispecific T cell engagers from
other multimeric species, which may have biological activities
other than the intended activity of the monomer. In one exemplary
purification scheme, a solution containing secreted polypeptides is
first subjected to a metal affinity chromatography, and
polypeptides are eluted with a gradient of imidazole
concentrations. This eluate is further purified using anion
exchange chromatography, and polypeptides are eluted using with a
gradient of sodium chloride concentrations. Finally, this eluate is
subjected to size exclusion chromatography to separate monomers
from multimeric species.
[0286] Antibodies with more than two valencies are contemplated.
For example, trispecific antibodies can be prepared. Tuft et al. J.
Immunol. 147: 60 (1991).
[0287] In certain embodiments, an antibody comprised in the
therapeutic agent may be further modified to contain additional
nonproteinaceous moieties that are known in the art and readily
available. The moieties suitable for derivatization of the antibody
include but are not limited to water soluble polymers. Non-limiting
examples of water soluble polymers include, but are not limited to,
polyethylene glycol (PEG), copolymers of ethylene glycol/propylene
glycol, carboxymethylcellulose, dextran, polyvinyl alcohol,
polyvinyl pyrrolidone, poly-1, 3-dioxolane, poly-1,3,6-trioxane,
ethylene/maleic anhydride copolymer, polyaminoacids (either
homopolymers or random copolymers), and dextran or poly(n-vinyl
pyrrolidone)polyethylene glycol, propropylene glycol homopolymers,
prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylated
polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.
Polyethylene glycol propionaldehyde may have advantages in
manufacturing due to its stability in water. The polymer may be of
any molecular weight, and may be branched or unbranched. The number
of polymers attached to the antibody may vary, and if more than one
polymer are attached, they can be the same or different molecules.
In general, the number and/or type of polymers used for
derivatization can be determined based on considerations including,
but not limited to, the particular properties or functions of the
antibody to be improved, whether the antibody derivative will be
used in a therapy under defined conditions, etc.
[0288] The therapeutic agent may also comprise an antibody
conjugated to one or more cytotoxic agents, such as
chemotherapeutic agents or drugs, growth inhibitory agents, toxins
(e.g., protein toxins, enzymatically active toxins of bacterial,
fungal, plant, or animal origin, or fragments thereof), or
radioactive isotopes.
[0289] In one embodiment, the therapeutic agent comprises an
antibody-drug conjugate (ADC) in which an antibody is conjugated to
one or more drugs, including but not limited to a maytansinoid (see
U.S. Pat. Nos. 5,208,020, 5,416,064 and European Patent EP 0 425
235 B1); an auristatin such as monomethylauristatin drug moieties
DE and DF (MMAE and MMAF) (see U.S. Pat. Nos. 5,635,483 and
5,780,588, and 7,498,298); a dolastatin; a calicheamicin or
derivative thereof (see U.S. Pat. Nos. 5,712,374, 5,714,586,
5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, and
5,877,296; Hinman et al., Cancer Res. 53:3336-3342 (1993); and Lode
et al., Cancer Res. 58:2925-2928 (1998)); an anthracycline such as
daunomycin or doxorubicin (see Kratz et al., Current Med. Chem.
13:477-523 (2006); Jeffrey et al., Bioorganic & Med. Chem.
Letters 16:358-362 (2006); Torgov et al., Bioconj. Chem. 16:717-721
(2005); Nagy et al., Proc. Natl. Acad. Sci. USA 97:829-834 (2000);
Dubowchik et al., Bioorg. & Med. Chem. Letters 12:1529-1532
(2002); King et al., J. Med. Chem. 45:4336-4343 (2002); and U.S.
Pat. No. 6,630,579); methotrexate; vindesine; a taxane such as
docetaxel, paclitaxel, larotaxel, tesetaxel, and ortataxel; a
trichothecene; and CC1065.
[0290] In another embodiment, the therapeutic agent comprises an
antibody as described herein conjugated to an enzymatically active
toxin or fragment thereof, including but not limited to diphtheria
A chain, nonbinding active fragments of diphtheria toxin, exotoxin
A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A
chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins,
dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and
PAP-S), Momordica charantia inhibitor, curcin, crotin, Saponaria
officinalis inhibitor, gelonin, mitogellin, restrictocin,
phenomycin, enomycin, and the tricothecenes.
[0291] In another embodiment, the therapeutic agent comprises an
antibody as described herein conjugated to a radioactive atom to
form a radioconjugate. A variety of radioactive isotopes are
available for the production of radioconjugates. Examples include
At.sup.211, I.sup.131, I.sup.125, Y.sup.90, Re.sup.186, Re.sup.188,
Sm.sup.153, Bi.sup.212, P.sup.32, Pb.sup.212 and radioactive
isotopes of Lu. When the radioconjugate is used for detection, it
may comprise a radioactive atom for scintigraphic studies, for
example Tc.sup.99m or I.sup.123, or a spin label for nuclear
magnetic resonance (NMR) imaging (also known as magnetic resonance
imaging, mri), such as iodine-123 again, iodine-131, indium-111,
fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium,
manganese or iron.
[0292] Conjugates of an antibody and cytotoxic agent may be made
using a variety of bifunctional protein coupling agents such as
N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),
succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate
(SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters
(such as dimethyl adipimidate HCl), active esters (such as
disuccinimidyl suberate), aldehydes (such as glutaraldehyde),
bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine),
bis-diazonium derivatives (such as
bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as
toluene 2,6-diisocyanate), and bis-active fluorine compounds (such
as 1,5-difluoro-2,4-dinitrobenzene). For example, a ricin
immunotoxin can be prepared as described in Vitetta et al., Science
238:1098 (1987). Carbon-14-labeled
1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid
(MX-DTPA) is an exemplary chelating agent for conjugation of
radionucleotide to the antibody. See WO94/11026. The linker may be
a "cleavable linker" facilitating release of a cytotoxic drug in
the cell. For example, an acid-labile linker, peptidase-sensitive
linker, photolabile linker, dimethyl linker or disulfide-containing
linker (Chari et al., Cancer Res. 52:127-131 (1992); U.S. Pat. No.
5,208,020) may be used.
[0293] In some embodiments, the therapeutic agent may comprise an
monoclonal antibody such as, but not limited to, alemtuzumab
(LEMTRADA.RTM.), bevacizumab (AVASTIN.RTM.), cetuximab
(ERBITUX.RTM.), panitumumab (VECTIBIX.RTM.), pertuzumab
(OMNITARG.RTM., 2C4), trastuzumab (HERCEPTIN.RTM.), tositumomab
(Bexxar.RTM.), abciximab (REOPRO.RTM.), adalimumab (HUMIRA.RTM.)
apolizumab, aselizumab, atlizumab, bapineuzumab, basiliximab
(SIMULECT.RTM.), bavituximab, belimumab (BENLYSTA.RTM.)
briankinumab, canakinumab (ILARIS.RTM.), cedelizumab, certolizumab
pegol (CIMZIA.RTM.), cidfusituzumab, cidtuzumab, cixutumumab,
clazakizumab, crenezumab, daclizumab (ZENAPAX.RTM.), dalotuzumab,
denosumab (PROLIA.RTM., XGEVA.RTM.), eculizumab (SOLIRIS.RTM.),
efalizumab, epratuzumab, erlizumab, felvizumab, fontolizumab,
golimumab (SIMPONI.RTM.), ipilimumab, imgatuzumab, infliximab
(REMICADE.RTM.), labetuzumab, lebrikizumab, lexatumumab,
lintuzumab, lucatumumab, lulizumab pegol, lumretuzumab,
mapatumumab, matuzumab, mepolizumab, mogamulizumab, motavizumab,
motovizumab, muronomab, natalizumab (TYSABRI.RTM.), necitumumab
(PORTRAZZA.RTM.), nimotuzumab (THERACIM.RTM.), nolovizumab,
numavizumab, olokizumab, omalizumab (XOLAIR.RTM.), onartuzumab
(also known as MetMAb), palivizumab (SYNAGIS.RTM.), pascolizumab,
pecfusituzumab, pectuzumab, pembrolizumab (KEYTRUDA.RTM.),
pexelizumab, priliximab, ralivizumab, ranibizumab (LUCENTIS.RTM.),
reslivizumab, reslizumab, resyvizumab, robatumumab, rontalizumab,
rovelizumab, ruplizumab, sarilumab, secukinumab, seribantumab,
sifalimumab, sibrotuzumab, siltuximab (SYLVANT.RTM.) siplizumab,
sontuzumab, tadocizumab, talizumab, tefibazumab, tocilizumab
(ACTEMRA.RTM.), toralizumab, tucusituzumab, umavizumab,
urtoxazumab, ustekinumab (STELARA.RTM.), vedolizumab
(ENTYVIO.RTM.), visilizumab, zanolimumab, zalutumumab.
[0294] In one embodiment, the therapeutic agent comprises an
antibody indicated for the treatment of cancer. In one embodiment,
the therapeutic agent comprises an antibody indicated for the
treatment of an autoimmune disease. In one embodiment, the
therapeutic agent is an immunotherapeutic agent. In one embodiment
the therapeutic agent is indicated for the treatment of cancer. In
some embodiments, in particular in relation aspects of the
invention concerned with the reduction of cytokine release
associated with the administration of a therapeutic agent in a
subject, the cancer is a B-cell proliferative disorder. In one
embodiment, the cancer is a CD20-positive B-cell proliferative
disorder. In one embodiment, the cancer is selected from the group
consisting of Non-Hodgkin lymphoma (NHL), acute lymphocytic
leukemia (ALL), chronic lymphocytic leukemia (CLL), diffuse large
B-cell lymphoma (DLBCL), follicular lymphoma (FL), mantle-cell
lymphoma (MCL), marginal zone lymphoma (MZL), Multiple myeloma
(MM), and Hodgkin lymphoma (HL). In one embodiment, the therapeutic
agent is an immunotherapeutic agent.
[0295] In one embodiment, the therapeutic agent is an
immunosuppressive agent. In one embodiment, the therapeutic agent
is indicated for the treatment of an autoimmune disease.
[0296] Without wishing to be bound to theory, it is thought that
enhancing T cell stimulation, by promoting an activating
co-stimulatory molecule or by inhibiting a negative co-stimulatory
molecule, may promote tumor cell death thereby treating or delaying
progression of cancer. In some embodiments, the therapeutic agent
may comprise an agonist directed against an activating
co-stimulatory molecule. In some embodiments, an activating
co-stimulatory molecule may include CD40, CD226, CD28, OX40, GITR,
CD137, CD27, MEM, or CD127. In some embodiments, the agonist
directed against an activating co-stimulatory molecule is an
agonist antibody that binds to CD40, CD226, CD28, OX40, CD137,
CD27, HVEM, or CD127. In some embodiments, the therapeutic agent
may comprise an antibody targeting GITR. In some embodiments, the
antibody targeting GITR is TRX518 some the therapeutic agent may
comprise an antagonist directed against an inhibitory
co-stimulatory molecule. In some embodiments, an inhibitory
co-stimulatory molecule may include CTLA-4 (also known as CD152),
PD-1, BTLA, VISTA, LAG-3, B7-H3, B7-H4, IDO, TIGIT, MICA/B, or
arginase. In some embodiments, the antagonist directed against an
inhibitory co-stimulatory molecule is an antagonist antibody that
binds to CTLA-4 PD-1, TIM-3, BTLA, VISTA, LAG-3, B7-H3, B7-H4, IDO,
TIGIT, MIC A/13, or arginase.
[0297] In some embodiments, the therapeutic agent may comprise an
anti-PD-1 antibody. In one embodiment the anti-PD-1 antibody is
selected from the group consisting of MDX-1106 (nivolumab), MK-3475
(pembrolizumab, formerly known as lambrolizumab), CT-011
(pidilizumab). MDX-1106, also known as MDX-1106-04, ONO-4538,
BMS-936558, or nivolumab, is an anti-PD-1 antibody described in
WO2006/121168. MK-3475, also known as pembrolizumab or (formerly)
lambrolizumab, is an anti-PD-1 antibody described in WO2009/114335.
CT-011, also known as hBAT, hBAT-1 or pidilizumab, is an anti-PID-1
antibody described in WO2009/101 611.
[0298] In some embodiments, the therapeutic agent may comprise an
immunoadhesin (e.g., an immunoadhesin comprising an extracellular
or PD-1 binding portion of PD-L1 or PD-L2 fused to a constant
region (e.g., an Fc region of an immunoglobulin sequence)). In one
embodiment, the therapeutic agent may comprise AMP-224, also known
as B7-DCIg (a PD-L2-Fc fusion soluble receptor described in
WO2010/027827 and WO2011/066342), In some embodiments, the
therapeutic agent may comprise an anti-PD-L1 antibody. In one
embodiment the anti-PD-L1 antibody is selected from the group
consisting of YW243.55.S70, MPDL3280A, MDX-1105, and MEDI4736.
Antibody YW243.55.S70 is an anti-PD-L1 antibody described in WO
2010/077634. MDS-1105, also known as BMS-936559, is an anti-PD-L1
antibody described in WO2007/005874. MEDI4736 is an anti-PD-L1
monoclonal antibody described in WO2011/066389 and US2013/034559.
In one embodiment, the anti-PD-L1 antibody is atezolizumab.
[0299] In some embodiments, the therapeutic agent may comprise an
antagonist directed against CTLA-4 (also known as CD152), for
example, a blocking antibody. In some embodiments, the therapeutic
agent may comprise ipilimumab (also known as MDX-010, MDX-101, or
YERVOY.RTM.). In some embodiments, the therapeutic agent may
comprise tremelimumab (also known as ticilimumab or CP-675,206). In
some embodiments, the therapeutic agent may comprise an antagonist
directed against B7-113 (also known as CD276), for example, a
blocking antibody. In some embodiments, the therapeutic agent may
comprise MGA271. In some embodiments, the therapeutic agent may
comprise an antagonist directed against a TGF beta, for example,
metelimumab (also known as CAT-192), fresolimumab (also known as
GC1008), or LY2157299.
[0300] In some embodiments, the therapeutic agent may comprise an
agonist directed against CD137 (also known as TNFRSF9, 4-1BB, or
ILA), for example, an activating antibody. In some embodiments, the
therapeutic agent may comprise urelumab (also known as BMS-663513).
In some embodiments, the therapeutic agent may comprise ligand of
CD137 (also known as TNFRSF9, 4-1BB, or ILA), such as 4-1BBL. In
some embodiments, the therapeutic agent may comprise an agonist
directed against CD40, for example, an activating antibody. In some
embodiments, the therapeutic agent may comprise CP-870893. In some
embodiments, the therapeutic agent may comprise an agonist directed
against OX40 (also known as CD134), for example, an activating
antibody. In some embodiments, the therapeutic agent may comprise
an anti-OX40 antibody (e.g., AgonOX). In some embodiments, the
therapeutic agent may comprise a ligand of OX40, such as OX40L. In
some embodiments, the therapeutic agent may comprise an agonist
directed against CD27, for example, an activating antibody. In some
embodiments, the therapeutic agent may comprise CDX-1127.
[0301] In some embodiments, the therapeutic agent may comprise a T
cell (e.g., a cytotoxic T cell or CTL) expressing a chimeric
antigen receptor (CAR). In some embodiments, the therapeutic agent
may comprise a T cell comprising a dominant-negative TGF beta
receptor, e.g, a dominant-negative TGF beta type II receptor.
[0302] In some embodiments, the therapeutic agent may comprise an
antibody-drug conjugate. In some embodiments, the antibody-drug
conjugate comprises mertansine or monomethyl auristatin E (MMAE).
In some embodiments, the therapeutic agent may comprise an
anti-NaPi2b antibody-MMAE conjugate (also known as DNIB0600A or
RG7599). In some embodiments, the therapeutic agent may comprise
trastuzumab emtansine (also known as T-DM1, ado-trastuzumab
emtansine, or KADCYLA.RTM.). In some embodiments, the therapeutic
agent may comprise DMUC5754A, In some embodiments, the therapeutic
agent may comprise an antibody-drug conjugate targeting the
endothelin B receptor (EDNBR), for example, an antibody directed
against EDNBR conjugated with MMAE (also known as DEDN6526A). In
some embodiments, the therapeutic agent may comprise gemtuzumab
ozogamicin (MYLOTARG.RTM.), In some embodiments, the therapeutic
agent may comprise inotuzumab ozogamicin. In some embodiments, the
therapeutic agent may comprise bivatuzumab mertansine. In some
embodiments, the therapeutic agent may comprise cantuzumab
mertansine. In some embodiments, the therapeutic agent may comprise
cantuzumab ravtansine. In some embodiments, the therapeutic agent
may comprise brentuximab vedotin (ADECTRIS.RTM.). In some
embodiments, the therapeutic agent may comprise pinatuzumab
vedotin. In some embodiments, the therapeutic agent may comprise
polatuzumab vedotin In some embodiments, the therapeutic agent may
comprise glembatumumab vedotin. In some embodiments, the
therapeutic agent may comprise lorvotuzumab mertansine. In some
embodiments, the therapeutic agent may comprise tacatuzumab
tetraxetan. In some embodiments, the therapeutic agent may comprise
vandortuzumab vedotin (DSTP3086S). In some embodiments, the
therapeutic agent may comprise ibritumomab tiuxetan
(ZEVALIN.RTM.)
[0303] In some embodiments, the therapeutic agent may comprise an
antibody directed against angiopoietin 2 (also known as Ang2). In
some embodiments, the therapeutic agent may comprise MEDI3617.
[0304] In some embodiments, the therapeutic agent may comprise an
antibody targeting CSF-1R (also known as M-CSFR or CD115). In some
embodiments, the therapeutic agent may comprise IMC-CS4
(LY3022855)). In some embodiments, the therapeutic agent may
comprise emactuzumab.
[0305] In some embodiment, the therapeutic agent may comprise a
cytokine. In some embodiments, the therapeutic agent may comprise
an interferon, for example interferon alpha or interferon gamma. In
some embodiments the therapeutic agent may comprise Roferon-A (also
known as recombinant Interferon alpha-2a). In some embodiments, the
therapeutic agent may comprise GM-CSF (also known as recombinant
human granulocyte macrophage colony stimulating factor, rhu GM-CSF,
sargramostim, or LEUKIN E.RTM.). In some embodiments, the
therapeutic agent may comprise aldesleukin (PROLEUKIN.RTM.). In
some embodiments, the therapeutic agent may comprise IL-12, In some
embodiments, the therapeutic agent may comprise IL-10.
[0306] In some embodiments, the therapeutic agent may comprise an
IL-2 fusion protein. In some embodiments, the therapeutic agent may
comprise tucotuzumab celmoleukin. In some embodiments, the
therapeutic agent may comprise darleukin. In some embodiments, the
therapeutic agent may comprise teleukin.
[0307] In some embodiments, the therapeutic agent may comprise an
IL-10 fusion protein. In some embodiments, the therapeutic agent
may comprise dekavil. In some embodiments, the therapeutic agent
may comprise a TNF fusion protein. In some embodiments, the
therapeutic agent may comprise fibromun.
[0308] In some embodiments, the therapeutic agent may comprise a
bispecific antibody. In some embodiments, the therapeutic agent may
comprise a bispecific antibody, such as, but not limited to,
duligotuzumab, MM-111, MM141, TF2, ABT-981, ABT-122, LY3164530,
SAR156597, GSK2434735, ozoralizumab, ALX-0761, ALX-0061, ALX-0141,
ACE910.
[0309] In some embodiments, the therapeutic agent may comprise a
bispecific antibody capable of binding to a T cell and a target
cell, e.g. a tumor cell. In some embodiment, the therapeutic agent
may comprise a bispecific antibody that specifically binds to CD3
on a T cell and to a target cell antigen. In some embodiment, the
therapeutic agent may comprise a bispecific T cell engager
(BITE.RTM.). In some embodiments, the therapeutic agent may
comprise a bispecific antibody directed against CD3 and CD19. In
one embodiment, the bispecific antibody is blinatumomab
(BLINCYTO.RTM.). In one embodiment, the bispecific antibody is
AFM11. In some embodiments, the therapeutic agent may comprise a
bispecific antibody directed against CD3 and EpCAM. In one
embodiment, the bispecific antibody is catumaxomab (REVOMAB.RTM.).
In one embodiment, the bispecific antibody is solitomab (AMG 110,
MT110). In some embodiments, the therapeutic agent may comprise a
bispecific antibody directed against CD3 and Her2. In one
embodiment, the bispecific antibody is ertumaxomab. In some
embodiments, the therapeutic agent may comprise a bispecific
antibody directed against CD3 and PSMA. In one embodiment, the
bispecific antibody is BAY2010112 (AMG212, MT112). In some
embodiments, the therapeutic agent may comprise a bispecific
antibody directed against CD3 and CEA. In one embodiment, the
bispecific antibody is MEDI565 (AMG211, MT111). In some
embodiments, the therapeutic agent may comprise a bispecific
antibody directed against CD3 and CD33. In one embodiment, the
bispecific antibody is AMG330. In some embodiments, the therapeutic
agent may comprise a bispecific antibody directed against CD3 and
CD123. In one embodiment, the bispecific antibody is MGD006. In one
embodiment, the bispecific antibody is XmAb.RTM. 14045. In some
embodiments, the therapeutic agent may comprise a bispecific
antibody directed against CD3 and CD38. In some embodiments, the
therapeutic agent may comprise a bispecific antibody directed
against CD3 and gpA33. In one embodiment, the bispecific antibody
is MGD007. In some embodiments, the therapeutic agent may comprise
a bispecific antibody directed against CD3 and CD20. In one
embodiment, the bispecific antibody is XmAb.RTM. 13676. In one
embodiment, the bispecific antibody is REGN1979. In one embodiment,
the bispecific antibody is FBTA05 (Lymphomun).
[0310] In some embodiments, the therapeutic agent may comprise a
bispecific antibody directed against CD30 and CD16A. In one
embodiment, the bispecific antibody is AFM13. In some embodiments,
the therapeutic agent may comprise a bispecific antibody directed
against DR5 and FAP. In some embodiments, the therapeutic agent may
comprise a bispecific antibody directed against Ang2 and VEGF. In
one embodiment, the bispecific antibody is vanucizumab.
[0311] In some embodiments, the therapeutic agent may comprise an
Fc domain. In some embodiments, the therapeutic agent may comprise
a fusion protein comprising an Fc domain.
[0312] In some embodiments, the therapeutic agent may comprise a
recombinant receptor or a fragment thereof. In some embodiments,
the receptor is a T cell receptor. In some embodiments, the
receptor is a TNF receptor. In some embodiments, the therapeutic
agent may comprise etanercept (ENBREL.RTM.). In some embodiments,
the receptor is a VEGF receptor. In some embodiments, the
therapeutic agent may comprise ziv-aflibercept (ZALTRAP.RTM.). In
some embodiments, the therapeutic agent may comprise aflibercept
(EYLEA.RTM.). In some embodiments, the receptor is an IL-1
receptor. In some embodiments, the therapeutic agent may comprise
rilonacept (ARCALYST.RTM.). In some embodiments, the therapeutic
agent may comprise IMCgp100. In some embodiments, the therapeutic
agent may comprise a chimeric antigen receptor (CAR). In some
embodiments, the therapeutic agent may comprise a Factor IX-Fc
fusion protein. In some embodiments, the therapeutic agent may
comprise a Factor VIII-Fc fusion protein. In some embodiments, the
therapeutic agent may comprise a CTLA-4-Fc fusion protein, such as
e.g. belatacept, abatacept (ORENCIA.RTM.). In one embodiment, the
therapeutic agent may comprise romiplostin.
[0313] In some embodiments, the therapeutic agent may comprise a
recombinant receptor ligand, such as a TNF receptor ligand.
[0314] In some embodiments, the therapeutic agent may comprise a
generic, biosimilar or non-comparable biologic version of an agent,
e.g. an antibody, named herein.
[0315] In one embodiment, the therapeutic agent does not comprise
obinutuzumab.
[0316] T Cell Activating Therapeutic Agents
[0317] The following describes in further detail T cell activating
therapeutic agents for which the invention may be useful, in
particular aspects of the invention concerned with the reduction of
cytokine release associated with the administration of a
therapeutic agent in a subject.
[0318] In some embodiments, the therapeutic agent comprises an
antibody that specifically binds to an activating T cell antigen.
In one embodiment, the therapeutic agent may comprise an antibody
that specifically binds to an antigen selected from the group of
CD3, CD28, CD137 (also known as 4-1BB), CD40, CD226, OX40, GITR,
CD27, HVEM, and CD127.
[0319] In one embodiment, the therapeutic agent comprises an
antibody that specifically binds to CD3, particularly
CD3.epsilon..
[0320] In one embodiment, the therapeutic agent comprises an
antibody that is or can compete for binding with antibody H2C (PCT
publication no. WO2008/119567), antibody V9 (Rodrigues et al., Int
J Cancer Suppl 7, 45-50 (1992) and U.S. Pat. No. 6,054,297),
antibody FN18 (Nooij et al., Eur J Immunol 19, 981-984 (1986)),
antibody SP34 (Pessano et al., EMBO J 4, 337-340 (1985)), antibody
OKT3 (Kung et al., Science 206, 347-349 (1979)), antibody WT31
(Spits et al., J Immunol 135, 1922 (1985)), antibody UCHT1 (Burns
et al., J Immunol 129, 1451-1457 (1982)), antibody 7D6 (Coulie et
al., Eur J Immunol 21, 1703-1709 (1991)) or antibody Leu-4. In some
embodiments, the therapeutic agent may also comprise an antibody
that specifically binds to CD3 as described in WO 2005/040220, WO
2005/118635, WO 2007/042261, WO 2008/119567, WO 2008/119565, WO
2012/162067, WO 2013/158856, WO 2013/188693, WO 2013/186613, WO
2014/110601, WO 2014/145806, WO 2014/191113, WO 2014/047231, WO
2015/095392, WO 2015/181098, WO 2015/001085, WO 2015/104346, WO
2015/172800, WO 2016/020444, or WO 2016/014974.
[0321] In one embodiment, the therapeutic agent may comprise an
antibody that specifically binds to a B-cell antigen, particularly
a malignant B-cell antigen. In one embodiment, the therapeutic
agent may comprise an antibody that specifically binds to an
antigen selected from the group consisting of CD20, CD19, CD22,
ROR-1, CD37 and CD5, particularly to CD20 or CD19.
[0322] In some embodiments, the therapeutic agent may comprise an
antibody selected from rituximab, ocrelizumab, ofatumumab,
ocaratuzumab, veltuzumab, and ublituximab.
[0323] In some embodiments, the therapeutic agent may comprise a
multispecific antibody, particularly a bispecific antibody. In some
embodiments, the therapeutic agent may comprise a bispecific
antibody capable of binding to a T cell and a target cell, e.g. a
tumor cell. In some embodiments, the target cell is a B-cell,
particularly a malignant B-cell. In some embodiments, the
therapeutic agent may comprise a bispecific antibody that
specifically binds to (i) an activating T cell antigen and (ii) a B
cell antigen. In some embodiments, the therapeutic agent may
comprise a bispecific antibody that specifically binds to CD3 on a
T cell and to a target cell antigen. In some embodiments, the
target cell antigen is a B-cell antigen, particularly a malignant
B-cell antigen. In some embodiments, the therapeutic agent may
comprise a bispecific T cell engager (BITE.RTM.).
[0324] In some embodiments, the therapeutic agent may comprise a
bispecific antibody directed against CD3 and CD20. In one
embodiment, the bispecific antibody is XmAb.RTM. 13676. In one
embodiment, the bispecific antibody is REGN1979. In one embodiment,
the bispecific antibody is FBTA05 (Lymphomun).
[0325] In some embodiments, the therapeutic agent may comprise a
bispecific antibody directed against CD3 and CD19. In one
embodiment, the bispecific antibody is blinatumomab
(BLINCYTO.RTM.). In one embodiment, the bispecific antibody is
AFM11. In one embodiment, the bispecific antibody is MGD011
(JNJ-64052781).
[0326] In some embodiments, the therapeutic agent may comprise a
bispecific antibody directed against CD3 and CD38. In one
embodiment, the bispecific antibody is XmAb.RTM. 13551, XmAb.RTM.
15426, or XmAb.RTM. 14702.
[0327] In some embodiments, the therapeutic agent may comprise a
bispecific antibody directed against CD3 and BCMA. In one
embodiment, the bispecific antibody is BI836909.
[0328] In some embodiments, the therapeutic agent may comprise a
bispecific antibody directed against CD3 and CD33. In one
embodiment, the bispecific antibody is AMG330.
[0329] In some embodiments, the therapeutic agent may comprise a
bispecific antibody directed against CD3 and CD123. In one
embodiment, the bispecific antibody is MGD006. In one embodiment,
the bispecific antibody is XmAb.RTM. 14045. In one embodiment, the
bispecific antibody is JNJ-63709178.
[0330] In some embodiments, the therapeutic agent may comprise a
recombinant receptor or a fragment thereof. In some embodiments,
the receptor is a T cell receptor (TCR). In some embodiments, the
therapeutic agent may comprise a chimeric antigen receptor
(CAR).
[0331] In some embodiments, the therapeutic agent may comprise a T
cell (e.g, a cytotoxic T cell or CTL) expressing a chimeric antigen
receptor (CAR). In some embodiments, the therapeutic agent may
comprise a T cell expressing a recombinant T cell receptor
(TCR).
[0332] In one embodiment, the therapeutic agent may comprise a CAR
that specifically binds to a B-cell antigen, particularly a
malignant B-cell antigen. In one embodiment, the therapeutic agent
may comprise a CAR that specifically binds to an antigen selected
from the group consisting of CD20, CD19, CD22, ROR-1, CD37 and CD5,
particularly to CD20 or CD19.
[0333] In some embodiments, the therapeutic agent may comprise a
CAR directed to CD19, or a T cell expressing a CAR directed to
CD19. In some embodiments, the therapeutic agent may comprise
KTE-C19, CTL019, JCAR-014, JCAR-015, JCAR-017, BPX-401,
UCART19,
[0334] In some embodiments, the therapeutic agent may comprise a
CAR directed to CD22, or a T cell expressing a CAR directed to
CD22. In some embodiments, the therapeutic agent may comprise
JCAR-018 or UCART22.
[0335] In some embodiments, the therapeutic agent may comprise an
agonist directed against an T cell activating co-stimulatory
molecule. In some embodiments, a T cell activating co-stimulatory
molecule may include CD40, CD226, CD28, OX40, GITR, CD137, CD27,
HVEM, or CD127. In some embodiments, the agonist directed against a
T cell activating co-stimulatory molecule is an agonist antibody
that binds to CD40, CD226, CD28, OX40, GITR, CD137, CD27, HVEM, or
CD127. In some embodiments, the therapeutic agent may comprise an
antibody targeting GITR. In some embodiments, the antibody
targeting GITR is TRX518.
[0336] In some embodiments, the therapeutic agent may comprise an
agonist directed against CD137 (also known as TNFRSF9, 4-1BB, or
ILA), for example, an activating antibody. In some embodiments, the
therapeutic agent may comprise urelumab (also known as BMS-663513).
In some embodiments, the therapeutic agent may comprise ligand of
CD137 (also known as TNFRSF9, 4-1BB, or ILA), such as 4-1BBL. In
some embodiments, the therapeutic agent may comprise an agonist
directed against CD40, for example, an activating antibody. In some
embodiments, the therapeutic agent may comprise CP-870893. In some
embodiments, the therapeutic agent may comprise an agonist directed
against OX40 (also known as CD134), for example, an activating
antibody. In some embodiments, the therapeutic agent may comprise
an anti-OX40 antibody (e.g., AgonOX). In some embodiments, the
therapeutic agent may comprise a ligand of OX40, such as OX40L. In
some embodiments, the therapeutic agent may comprise an agonist
directed against CD27, for example, an activating antibody. In some
embodiments, the therapeutic agent may comprise CDX-1127.
[0337] Particular Therapeutic Agents
[0338] (i) Reduction of the Formation of Anti-Drug Antibodies
(ADAs)
[0339] The therapeutic agents described in the following are
particularly useful in the invention, in particular in relation
aspects of the invention concerned with the reduction of the
formation of anti-drug antibodies (ADAs) against a therapeutic
agent in a subject.
[0340] In some embodiments, the therapeutic agent comprises an
antibody that specifically binds to carcinoembryonic antigen
(CEA).
[0341] In one embodiment, the antibody that specifically binds to
CEA comprises a heavy chain variable region comprising the heavy
chain CDR (HCDR) 1 of SEQ ID NO: 14, the HCDR2 of SEQ ID NO: 15,
and the HCDR3 of SEQ ID NO: 16; and a light chain variable region
comprising the light chain CDR (LCDR) 1 of SEQ ID NO: 17, the LCDR2
of SEQ ID NO: 18 and the LCDR3 of SEQ ID NO: 19. In a further
embodiment, the antibody that specifically binds CEA comprises a
heavy chain variable region sequence that is at least 80%, 85%,
90%, 95%, 96%, 97%, 98%, or 99% identical to of SEQ ID NO: 20 and a
light chain variable region sequence that is at least 80%, 85%,
90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID
NO: 21. In a further embodiment, the antibody that specifically
binds CEA comprises the heavy chain variable region sequence of SEQ
ID NO: 20 and the light chain variable region sequence of SEQ ID
NO: 21.
[0342] In one embodiment, the antibody that specifically binds to
CEA comprises a heavy chain variable region comprising the heavy
chain CDR (HCDR) 1 of SEQ ID NO: 136, the HCDR2 of SEQ ID NO: 137,
and the HCDR3 of SEQ ID NO: 138; and a light chain variable region
comprising the light chain CDR (LCDR) 1 of SEQ ID NO: 139, the
LCDR2 of SEQ ID NO: 140 and the LCDR3 of SEQ ID NO: 141. In a
further embodiment, the antibody that specifically binds CEA
comprises a heavy chain variable region sequence that is at least
80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to of SEQ ID
NO: 142 and a light chain variable region sequence that is at least
80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence
of SEQ ID NO: 143. In a further embodiment, the antibody that
specifically binds CEA comprises the heavy chain variable region
sequence of SEQ ID NO: 142 and the light chain variable region
sequence of SEQ ID NO: 143.
[0343] In one embodiment, the antibody that specifically binds to
CEA is a full-length antibody. In one embodiment, the antibody that
specifically binds to CEA is an antibody of the human IgG class,
particularly an antibody of the human IgG.sub.1 class. In one
embodiment, the antibody that specifically binds to CEA is an
antibody fragment, particularly a Fab molecule or a scFv molecule,
more particularly a Fab molecule. In one embodiment, the antibody
that specifically binds to CEA is a humanized antibody.
[0344] In some embodiments, the therapeutic agent comprises an
antibody that specifically binds to fibroblast activation protein
(FAP). In one embodiment, the antibody that specifically binds FAP
comprises a heavy chain variable region sequence that is at least
80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to of SEQ ID
NO: 25 and a light chain variable region sequence that is at least
80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence
of SEQ ID NO: 26. In a further embodiment, the antibody that
specifically binds FAP comprises the heavy chain variable region
sequence of SEQ ID NO: 25 and the light chain variable region
sequence of SEQ ID NO: 26.
[0345] In one embodiment, the antibody that specifically binds to
FAP is a full-length antibody. In one embodiment, the antibody that
specifically binds to FAP is an antibody of the human IgG class,
particularly an antibody of the human IgG.sub.1 class. In one
embodiment, the antibody that specifically binds to FAP is an
antibody fragment, particularly a Fab molecule or a scFv molecule,
more particularly a Fab molecule. In one embodiment, the antibody
that specifically binds to FAP is a human antibody.
[0346] In some embodiments, the therapeutic agent comprises an
antibody that specifically binds to CD3, particularly CD3 epsilon.
In one embodiment, the antibody that specifically binds to CD3
comprises a heavy chain variable region comprising the heavy chain
CDR (HCDR) 1 of SEQ ID NO: 32, the HCDR2 of SEQ ID NO: 33, and the
HCDR3 of SEQ ID NO: 34; and a light chain variable region
comprising the light chain CDR (LCDR) 1 of SEQ ID NO: 35, the LCDR2
of SEQ ID NO: 36 and the LCDR3 of SEQ ID NO: 37. In a further
embodiment, the antibody that specifically binds CD3 comprises a
heavy chain variable region sequence that is at least 80%, 85%,
90%, 95%, 96%, 97%, 98%, or 99% identical to of SEQ ID NO: 38 and a
light chain variable region sequence that is at least 80%, 85%,
90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID
NO: 39. In a further embodiment, the antibody that specifically
binds CD3 comprises the heavy chain variable region sequence of SEQ
ID NO: 38 and the light chain variable region sequence of SEQ ID
NO: 39.
[0347] In one embodiment, the antibody that specifically binds to
CD3 is a full-length antibody. In one embodiment, the antibody that
specifically binds to CD3 is an antibody of the human IgG class,
particularly an antibody of the human IgG.sub.1 class. In one
embodiment, the antibody that specifically binds to CD3 is an
antibody fragment, particularly a Fab molecule or a scFv molecule,
more particularly a Fab molecule. In a particular embodiment, the
antibody that specifically binds to CD3 is a crossover Fab molecule
wherein the variable domains or the constant domains of the Fab
heavy and light chain are exchanged (i.e. replaced by each other).
In one embodiment, the antibody that specifically binds to CD3 is a
humanized antibody.
[0348] In some embodiments, the therapeutic agent comprises a
cytokine. In one embodiment the cytokine is selected from the group
consisting of, GM-CSF, IL-1.alpha., IL-1.beta., IL-2, IL-3, IL-4,
IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-15, IFN-.alpha.,
IFN-.gamma., MIP-1.alpha., MIP-1.beta., TGF-.beta., TNF-.alpha.,
and TNF-.beta.. In one embodiment, the cytokine is IL-2,
particularly human IL-2. The sequence of wild-type human IL-2 is
shown in SEQ ID NO: 12.
[0349] In one embodiment, the therapeutic agent comprises a mutant
IL-2 polypeptide having reduced binding affinity to the
.alpha.-subunit of the IL-2 receptor as compared to wild-type IL-2.
Together with the .beta.- and .gamma.-subunits (also known as CD122
and CD132, respectively), the .alpha.-subunit (also known as CD25)
forms the heterotrimeric high-affinity IL-2 receptor, while the
dimeric receptor consisting only of the .beta.- and
.gamma.-subunits is termed the intermediate-affinity IL-2 receptor.
A mutant IL-2 polypeptide with reduced binding to the
.alpha.-subunit of the IL-2 receptor has a reduced ability to
induce IL-2 signaling in regulatory T (T.sub.reg) cells, induces
less activation-induced cell death (AICD) in T cells, and has a
reduced toxicity profile in vivo, compared to a wild-type IL-2
polypeptide (see e.g. WO 2012/107417, incorporated herein by
reference in its entirety).
[0350] In a more specific embodiment, the mutant IL-2 polypeptide
comprises three amino acid substitutions at the positions
corresponding to residue 42, 45 and 72 of human IL-2. In an even
more specific embodiment, the mutant IL-2 polypeptide is a human
IL-2 polypeptide comprising the amino acid substitutions F42A, Y45A
and L72G (numbering relative to the human IL-2 sequence SEQ ID NO:
12). In one embodiment the mutant IL-2 polypeptide additionally
comprises an amino acid mutation at a position corresponding to
position 3 of human IL-2, which eliminates the O-glycosylation site
of IL-2. In one embodiment said amino acid mutation which
eliminates the O-glycosylation site of IL-2 at a position
corresponding to residue 3 of human IL-2 is an amino acid
substitution selected from the group of T3A, T3G, T3Q, T3E, T3N,
T3D, T3R, T3K, and T3P. Particularly, said additional amino acid
mutation is an amino acid substitution replacing a threonine
residue by an alanine residue. A particular mutant IL-2 polypeptide
useful in the invention comprises four amino acid substitutions at
positions corresponding to residues 3, 42, 45 and 72 of human IL-2.
Specific amino acid substitutions are T3A, F42A, Y45A and L72G.
This mutant IL-2 polypeptide exhibits no detectable binding to
CD25, reduced ability to induce apoptosis in T cells, reduced
ability to induce IL-2 signaling in T.sub.reg cells, and a reduced
toxicity profile in vivo (see e.g. WO 2012/107417, incorporated
herein by reference in its entirety). However, it retains ability
to activate IL-2 signaling in effector cells, to induce
proliferation of effector cells, and to generate IFN-.gamma. as a
secondary cytokine by NK cells.
[0351] The IL-2 or mutant IL-2 polypeptide according to any of the
above embodiments may comprise additional mutations that provide
further advantages such as increased expression or stability. For
example, the cysteine at position 125 may be replaced with a
neutral amino acid such as serine, alanine, threonine or valine,
yielding C125S IL-2, C125A IL-2, C125T IL-2 or C125V IL-2
respectively, as described in U.S. Pat. No. 4,518,584. As described
therein, one may also delete the N-terminal alanine residue of IL-2
yielding such mutants as des-A1 C125S or des-A1 C125A.
Alternatively or conjunctively, the IL-2 mutant may include a
mutation whereby methionine normally occurring at position 104 of
wild-type human IL-2 is replaced by a neutral amino acid such as
alanine (see U.S. Pat. No. 5,206,344). The resulting mutants, e.
g., des-A1 M104A IL-2, des-A1 M104A C125S IL-2, M104A IL-2, M104A
C125A IL-2, des-A1 M104A C125A IL-2, or M104A C125S IL-2 (these and
other mutants may be found in U.S. Pat. No. 5,116,943 and in Weiger
et al., Eur J Biochem 180, 295-300 (1989)) may be used in
conjunction with the particular IL-2 mutations described
herein.
[0352] Thus, in certain embodiments the IL-2 or mutant IL-2
polypeptide comprises an additional amino acid mutation at a
position corresponding to residue 125 of human IL-2. In one
embodiment said additional amino acid mutation is the amino acid
substitution C125A.
[0353] In certain embodiments the mutant IL-2 polypeptide is
essentially a full-length IL-2 molecule, particularly a human
full-length IL-2 molecule. In one embodiment, the mutant IL-2
polypeptide comprises a polypeptide sequence that is at least 80%,
at least 85%, or at least 90% identical to the sequence of SEQ ID
NO: 12.
[0354] In a specific embodiment the mutant IL-2 polypeptide
comprises the polypeptide sequence of SEQ ID NO: 13.
[0355] In some embodiments, the therapeutic agent comprises an
immunoconjugate. Particular immunoconjugates are described in WO
2012/107417 and WO 2012/146628 (each incorporated herein by
reference in its entirety).
[0356] In one embodiment, the immunoconjugate comprises an antibody
that specifically binds to CEA as described herein, and a mutant
IL-2 polypeptide as described herein. In one embodiment, the
antibody is a full-length antibody.
[0357] In one embodiment the therapeutic agent comprises an
immunoconjugate comprising [0358] (i) an antibody of the human
IgG.sub.1 subclass that specifically binds to CEA and comprises a
heavy chain variable region comprising the heavy chain CDR (HCDR) 1
of SEQ ID NO: 14, the HCDR2 of SEQ ID NO: 15, and the HCDR3 of SEQ
ID NO: 16; and a light chain variable region comprising the light
chain CDR (LCDR) 1 of SEQ ID NO: 17, the LCDR2 of SEQ ID NO: 18 and
the LCDR3 of SEQ ID NO: 19; and [0359] (ii) a mutant human IL-2
polypeptide comprising the amino acid substitutions F42A, Y45A and
L72G (numbering relative to the human IL-2 sequence SEQ ID NO:
12).
[0360] In one embodiment, the immunoconjugate comprises an antibody
that specifically binds to FAP as described herein, and a mutant
IL-2 polypeptide as described herein. In one embodiment, the
antibody is a full-length antibody.
[0361] In one embodiment the therapeutic agent comprises an
immunoconjugate comprising [0362] (i) an antibody of the human
IgG.sub.1 subclass that specifically binds to FAP and comprises the
heavy chain variable region of SEQ ID NO: 25; and the light chain
variable region of SEQ ID NO: 26; and [0363] (ii) a mutant human
IL-2 polypeptide comprising the amino acid substitutions F42A, Y45A
and L72G (numbering relative to the human IL-2 sequence SEQ ID NO:
12).
[0364] In one embodiment, the immunoconjugate comprises no more
than one mutant IL-2 polypeptide. In one embodiment, the mutant
IL-2 polypeptide is fused to the carboxy-terminal amino acid of one
of the antibody heavy chains, optionally through a linker peptide.
Suitable, non-immunogenic linker peptides include, for example,
(G.sub.4S).sub.n, (SG.sub.4).sub.n or G.sub.4(SG.sub.4).sub.n
linker peptides, wherein n is generally a number between 1 and 10,
typically between 2 and 4. In one embodiment, the linker peptide is
(G.sub.4S).sub.3.
[0365] In one embodiment, the immunoconjugate comprises a
polypeptide comprising a sequence that is at least 80%, 85%, 90%,
95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO:
22, a polypeptide comprising a sequence that is at least 80%, 85%,
90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID
NO: 23, and a polypeptide comprising a sequence that is at least
80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence
of SEQ ID NO: 24.
[0366] In one embodiment, the immunoconjugate comprises a
polypeptide comprising the sequence of SEQ ID NO: 22, a polypeptide
comprising the sequence of SEQ ID NO: 23, and a polypeptide
comprising the sequence of SEQ ID NO: 24.
[0367] In one embodiment, the immunoconjugate is cergutuzumab
amunaleukin (see WHO Drug Information (International Nonproprietary
Names for Pharmaceutical Substances), Recommended INN: List 75,
2016, pre-publication copy" (incorporated herein by reference in
its entirety).
[0368] In one embodiment, the immunoconjugate comprises a
polypeptide comprising a sequence that is at least 80%, 85%, 90%,
95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO:
27, a polypeptide comprising a sequence that is at least 80%, 85%,
90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID
NO: 28, and a polypeptide comprising a sequence that is at least
80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence
of SEQ ID NO: 29.
[0369] In one embodiment, the immunoconjugate comprises a
polypeptide comprising the sequence of SEQ ID NO: 27, a polypeptide
comprising the sequence of SEQ ID NO: 28, and a polypeptide
comprising the sequence of SEQ ID NO: 29.
[0370] In one embodiment, the therapeutic agent comprises a
bispecific antibody. Particular bispecific antibodies are described
in PCT publication nos. WO 2013/026833 and WO 2014/131712 and in
PCT application no. PCT/EP2016/073171 (each incorporated herein by
reference in its entirety).
[0371] In one embodiment, the bispecific antibody comprises an
antibody that specifically binds to CEA as described herein, and an
antibody that specifically binds to CD3 as described herein. In one
embodiment, the bispecific antibody comprises a first antibody that
specifically binds to CD3 as described herein, and a second and a
third antibody that specifically bind to CEA as described herein.
In one embodiment, the first antibody is a crossover Fab molecule
as described herein, and the second and the first antibody are each
a conventional Fab molecule. In one embodiment, the bispecific
antibody further comprises an Fc domain as described herein. The
bispecific antibody may have the antibody formats described herein
and may comprise the antigen binding moieties described herein. The
bispecific antibody may comprise modifications in the Fc region
and/or the antigen binding moieties as described herein.
[0372] In one embodiment the therapeutic agent comprises a
bispecific antibody comprising
[0373] (i) a first antigen binding moiety that specifically binds
to CD3, comprising a heavy chain variable region comprising the
heavy chain CDR (HCDR) 1 of SEQ ID NO: 32, the HCDR2 of SEQ ID NO:
33, and the HCDR3 of SEQ ID NO: 34; and a light chain variable
region comprising the light chain CDR (LCDR) 1 of SEQ ID NO: 35,
the LCDR2 of SEQ ID NO: 36 and the LCDR3 of SEQ ID NO: 37, wherein
the first antigen binding moiety is a crossover Fab molecule
wherein either the variable or the constant regions, particularly
the constant regions, of the Fab light chain and the Fab heavy
chain are exchanged;
[0374] (ii) a second and a third antigen binding moiety that
specifically bind to CEA, comprising a heavy chain variable region
comprising the heavy chain CDR (HCDR) 1 of SEQ ID NO: 14, the HCDR2
of SEQ ID NO: 15, and the HCDR3 of SEQ ID NO: 16; and a light chain
variable region comprising the light chain CDR (LCDR) 1 of SEQ ID
NO: 17, the LCDR2 of SEQ ID NO: 18 and the LCDR3 of SEQ ID NO: 19,
wherein the second and third antigen binding moiety are each a Fab
molecule, particularly a conventional Fab molecule;
[0375] (iii) an Fc domain composed of a first and a second subunit
capable of stable association, wherein the second antigen binding
moiety is fused at the C-terminus of the Fab heavy chain to the
N-terminus of the Fab heavy chain of the first antigen binding
moiety, and the first antigen binding moiety is fused at the
C-terminus of the Fab heavy chain to the N-terminus of the first
subunit of the Fc domain, and wherein the third antigen binding
moiety is fused at the C-terminus of the Fab heavy chain to the
N-terminus of the second subunit of the Fc domain.
[0376] In one embodiment, the first antigen binding moiety that
specifically binds to CD3, comprises the heavy chain variable
region of SEQ ID NO: 38, and the light chain variable region of SEQ
ID NO: 39. In one embodiment, the second and third antigen binding
moieties that specifically bind to CEA comprise the heavy chain
variable region of SEQ ID NO: 20, and the light chain variable
region of SEQ ID NO: 21.
[0377] In one embodiment, the antigen binding moieties and the Fc
region are fused to each other by peptide linkers, particularly by
peptide linkers as in SEQ ID NO: 42 and SEQ ID NO: 43. In one
embodiment, the bispecific antibody comprises a polypeptide
comprising a sequence that is at least 80%, 85%, 90%, 95%, 96%,
97%, 98%, or 99% identical to the sequence of SEQ ID NO: 40, a
polypeptide comprising a sequence that is at least 80%, 85%, 90%,
95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO:
41, a polypeptide comprising a sequence that is at least 80%, 85%,
90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID
NO: 42, and a polypeptide comprising a sequence that is at least
80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence
of SEQ ID NO: 43. In one embodiment, the bispecific antibody
comprises a polypeptide comprising the sequence of SEQ ID NO: 40, a
polypeptide comprising the sequence of SEQ ID NO: 41, a polypeptide
comprising the sequence of SEQ ID NO: 42, and a polypeptide
comprising the sequence of SEQ ID NO: 43. (CEA TCB)
[0378] In one embodiment the therapeutic antibody comprises a
bispecific antibody comprising
[0379] (i) a first antigen binding moiety that specifically binds
to CD3, comprising a heavy chain variable region comprising the
heavy chain CDR (HCDR) 1 of SEQ ID NO: 32, the HCDR2 of SEQ ID NO:
33, and the HCDR3 of SEQ ID NO: 34; and a light chain variable
region comprising the light chain CDR (LCDR) 1 of SEQ ID NO: 35,
the LCDR2 of SEQ ID NO: 36 and the LCDR3 of SEQ ID NO: 37, wherein
the first antigen binding moiety is a crossover Fab molecule
wherein either the variable or the constant regions, particularly
the variable regions, of the Fab light chain and the Fab heavy
chain are exchanged;
[0380] (ii) a second and a third antigen binding moiety that
specifically bind to CEA, comprising a heavy chain variable region
comprising the heavy chain CDR (HCDR) 1 of SEQ ID NO: 136, the
HCDR2 of SEQ ID NO: 137, and the HCDR3 of SEQ ID NO: 138; and a
light chain variable region comprising the light chain CDR (LCDR) 1
of SEQ ID NO: 139, the LCDR2 of SEQ ID NO: 140 and the LCDR3 of SEQ
ID NO: 141, wherein the second and third antigen binding moiety are
each a Fab molecule, particularly a conventional Fab molecule;
[0381] (iii) an Fc domain composed of a first and a second subunit
capable of stable association, wherein the second antigen binding
moiety is fused at the C-terminus of the Fab heavy chain to the
N-terminus of the Fab heavy chain of the first antigen binding
moiety, and the first antigen binding moiety is fused at the
C-terminus of the Fab heavy chain to the N-terminus of the first
subunit of the Fc domain, and wherein the third antigen binding
moiety is fused at the C-terminus of the Fab heavy chain to the
N-terminus of the second subunit of the Fc domain.
[0382] In one embodiment, the first antigen binding moiety that
specifically binds to CD3, comprises the heavy chain variable
region of SEQ ID NO: 38, and the light chain variable region of SEQ
ID NO: 39. In one embodiment, the second and third antigen binding
moiety that specifically bind to CEA comprise the heavy chain
variable region of SEQ ID NO: 142, and the light chain variable
region of SEQ ID NO: 143.
[0383] In one embodiment, the antigen binding moieties and the Fc
region are fused to each other by peptide linkers, particularly by
peptide linkers as in SEQ ID NO: 145 and SEQ ID NO: 146.
[0384] In one embodiment, in the constant domain CL of the second
and the third Fab molecule under (ii) the amino acid at position
124 is substituted by lysine (K) (numbering according to Kabat) and
the amino acid at position 123 is substituted by lysine (K) or
arginine (R), particularly by arginine (R) (numbering according to
Kabat), and in the constant domain CH1 of the second and the third
Fab molecule under (ii) the amino acid at position 147 is
substituted by glutamic acid (E) (numbering according to Kabat EU
index) and the amino acid at position 213 is substituted by
glutamic acid (E) (numbering according to Kabat EU index).
[0385] In one embodiment, the bispecific antibody comprises a
polypeptide comprising a sequence that is at least 80%, 85%, 90%,
95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO:
144, a polypeptide comprising a sequence that is at least 80%, 85%,
90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID
NO: 145, a polypeptide comprising a sequence that is at least 80%,
85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of
SEQ ID NO: 146, and a polypeptide comprising a sequence that is at
least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the
sequence of SEQ ID NO: 147.
[0386] In one embodiment, the bispecific antibody comprises a
polypeptide comprising the sequence of SEQ ID NO: 144, a
polypeptide comprising the sequence of SEQ ID NO: 145, a
polypeptide comprising the sequence of SEQ ID NO: 146, and a
polypeptide comprising the sequence of SEQ ID NO: 147.
[0387] (ii) Reduction of Cytokine Release
[0388] The therapeutic agents described in the following are
particularly useful in the invention, in particular in relation
aspects of the invention concerned with the reduction of cytokine
release associated with the administration of a therapeutic agent
in a subject.
[0389] The aspects of the invention concerned with the reduction of
cytokine release associated with the administration of a
therapeutic agent in a subject are particularly useful in
connection with therapeutic agents that are activating T-cells in
the subject (T cell activating therapeutic agents), i.e. have the
ability of inducing T-cell activation in the subject. Such
therapeutic agents include, for example, antibodies directed to
T-cell antigens (particularly activating T-cell antigens), or
T-cells modified with chimeric antigen receptors (CAR) or
recombinant T-cell receptors (TCR). The aspects of the invention
concerned with the reduction of cytokine release associated with
the administration of a therapeutic agent in a subject are
particularly useful in connection with B-cell targeted T-cell
activating therapeutic agents.
[0390] In some embodiments, the therapeutic agent comprises an
antibody that specifically binds to CD3, particularly CD3
epsilon.
[0391] In one embodiment, the antibody that specifically binds to
CD3 comprises a heavy chain variable region comprising the heavy
chain CDR (HCDR) 1 of SEQ ID NO: 32, the HCDR2 of SEQ ID NO: 33,
and the HCDR3 of SEQ ID NO: 34; and a light chain variable region
comprising the light chain CDR (LCDR) 1 of SEQ ID NO: 35, the LCDR2
of SEQ ID NO: 36 and the LCDR3 of SEQ ID NO: 37. In a further
embodiment, the antibody that specifically binds CD3 comprises a
heavy chain variable region sequence that is at least 80%, 85%,
90%, 95%, 96%, 97%, 98%, or 99% identical to of SEQ ID NO: 38 and a
light chain variable region sequence that is at least 80%, 85%,
90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID
NO: 39. In a further embodiment, the antibody that specifically
binds CD3 comprises the heavy chain variable region sequence of SEQ
ID NO: 38 and the light chain variable region sequence of SEQ ID
NO: 39.
[0392] In one embodiment, the antibody that specifically binds to
CD3 comprises a heavy chain variable region comprising the heavy
chain HVR 1 (H1-HVR) of SEQ ID NO: 120, the H2-HVR of SEQ ID NO:
121, and the H3-HVR of SEQ ID NO: 122; and a light chain variable
region comprising the light chain HVR 1 (L1-HVR) of SEQ ID NO: 123,
the L2-HVR of SEQ ID NO: 124 and the L3-HVR of SEQ ID NO: 125. In a
further embodiment, the antibody that specifically binds CD3
comprises a heavy chain variable region sequence that is at least
80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to of SEQ ID
NO: 126 and a light chain variable region sequence that is at least
80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence
of SEQ ID NO: 127. In a further embodiment, the antibody that
specifically binds CD3 comprises the heavy chain variable region
sequence of SEQ ID NO: 126 and the light chain variable region
sequence of SEQ ID NO: 127.
[0393] In one embodiment, the antibody that specifically binds to
CD3 is a full-length antibody. In one embodiment, the antibody that
specifically binds to CD3 is an antibody of the human IgG class,
particularly an antibody of the human IgG.sub.1 class. In one
embodiment, the antibody that specifically binds to CD3 is an
antibody fragment, particularly a Fab molecule or a scFv molecule,
more particularly a Fab molecule. In a particular embodiment, the
antibody that specifically binds to CD3 is a crossover Fab molecule
wherein the variable domains or the constant domains of the Fab
heavy and light chain are exchanged (i.e. replaced by each other).
In one embodiment, the antibody that specifically binds to CD3 is a
humanized antibody.
[0394] In one embodiment, the therapeutic agent comprises a
multispecific antibody, particularly a bispecific antibody. In one
embodiment, the multispecific antibody specifically binds to (i) an
activating T cell antigen and (ii) a B cell antigen. Particular
bispecific antibodies are described in PCT publication no. WO
2016/020309 and PCT application no. PCT/EP2016/073041, as well as
PCT publication no. WO 2015/095392 (each incorporated herein by
reference in its entirety).
[0395] In one embodiment, the bispecific antibody specifically
binds to CD3 and CD20. In one embodiment, the bispecific antibody
comprises an antigen binding moiety that specifically binds to
CD20, and an antigen binding moiety that specifically binds to CD3.
In one embodiment, the bispecific antibody comprises a first
antigen binding moiety that specifically binds to CD3, and a second
and a third antigen binding moiety that specifically bind to CD20.
In one embodiment, the first antigen binding moiety is a crossover
Fab molecule, and the second and the first antigen binding moiety
are each a conventional Fab molecule. In one embodiment, the
bispecific antibody further comprises an Fc domain. The bispecific
antibody may have the antibody formats described herein and may
comprise the antigen binding moieties described herein. The
bispecific antibody may comprise modifications in the Fc region
and/or the antigen binding moieties as described herein.
[0396] In one embodiment, the therapeutic agent comprises a
bispecific antibody comprising
[0397] (i) an antigen binding moiety that specifically binds to CD3
and comprises a heavy chain variable region comprising the heavy
chain CDR (HCDR) 1 of SEQ ID NO: 32, the HCDR2 of SEQ ID NO: 33,
and the HCDR3 of SEQ ID NO: 34; and a light chain variable region
comprising the light chain CDR (LCDR) 1 of SEQ ID NO: 35, the LCDR2
of SEQ ID NO: 36 and the LCDR3 of SEQ ID NO: 37; and
[0398] (ii) an antigen binding moiety that specifically binds to
CD20 and comprises a heavy chain variable region comprising the
heavy chain CDR (HCDR) 1 of SEQ ID NO: 4, the HCDR2 of SEQ ID NO:
5, and the HCDR3 of SEQ ID NO: 6; and a light chain variable region
comprising the light chain CDR (LCDR) 1 of SEQ ID NO: 7, the LCDR2
of SEQ ID NO: 8 and the LCDR3 of SEQ ID NO: 9.
[0399] In one embodiment, the therapeutic agent comprises a
bispecific antibody comprising
[0400] (i) an antigen binding moiety that specifically binds to CD3
and comprises a heavy chain variable region of SEQ ID NO: 38; and a
light chain variable region of SEQ ID NO: 39; and
[0401] (ii) an antigen binding moiety that specifically binds to
CD20 and comprises a heavy chain variable region of SEQ ID NO: 10;
and a light chain variable region of SEQ ID NO: 11.
[0402] In a particular embodiment, the therapeutic agent comprises
a bispecific antibody comprising
[0403] a) a first Fab molecule which specifically binds to a first
antigen;
[0404] b) a second Fab molecule which specifically binds to a
second antigen, and wherein the variable domains VL and VH of the
Fab light chain and the Fab heavy chain are replaced by each
other;
[0405] c) a third Fab molecule which specifically binds to the
first antigen; and
[0406] d) an Fc domain composed of a first and a second subunit
capable of stable association; wherein
[0407] (i) the first antigen is CD20 and the second antigen is CD3,
particularly CD3 epsilon;
[0408] (ii) the first Fab molecule under a) and the third Fab
molecule under c) each comprise the heavy chain complementarity
determining region (CDR) 1 of SEQ ID NO: 4, the heavy chain CDR 2
of SEQ ID NO: 5, the heavy chain CDR 3 of SEQ ID NO: 6, the light
chain CDR 1 of SEQ ID NO: 7, the light chain CDR 2 of SEQ ID NO: 8
and the light chain CDR 3 of SEQ ID NO: 9, and the second Fab
molecule under b) comprises the heavy chain CDR 1 of SEQ ID NO: 32,
the heavy chain CDR 2 of SEQ ID NO: 33, the heavy chain CDR 3 of
SEQ ID NO: 34, the light chain CDR 1 of SEQ ID NO: 35, the light
chain CDR 2 of SEQ ID NO: 36 and the light chain CDR 3 of SEQ ID
NO: 37;
[0409] (iii) in the constant domain CL of the first Fab molecule
under a) and the third Fab molecule under c) the amino acid at
position 124 is substituted by lysine (K) (numbering according to
Kabat) and the amino acid at position 123 is substituted by lysine
(K) or arginine (R), particularly by arginine (R) (numbering
according to Kabat), and wherein in the constant domain CH1 of the
first Fab molecule under a) and the third Fab molecule under c) the
amino acid at position 147 is substituted by glutamic acid (E)
(numbering according to Kabat EU index) and the amino acid at
position 213 is substituted by glutamic acid (E) (numbering
according to Kabat EU index); and
[0410] (iv) the first Fab molecule under a) is fused at the
C-terminus of the Fab heavy chain to the N-terminus of the Fab
heavy chain of the second Fab molecule under b), and the second Fab
molecule under b) and the third Fab molecule under c) are each
fused at the C-terminus of the Fab heavy chain to the N-terminus of
one of the subunits of the Fc domain under d).
[0411] In one embodiment, the first Fab molecule under a) and the
third Fab molecule under c) each comprise a heavy chain variable
region that is at least 95%, 96%, 97%, 98%, or 99% identical to the
sequence of SEQ ID NO: 10, and a light chain variable region that
is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of
SEQ ID NO: 11.
[0412] In one embodiment, the first Fab molecule under a) and the
third Fab molecule under c) each comprise the heavy chain variable
region sequence of SEQ ID NO: 10, and the light chain variable
region sequence of SEQ ID NO: 11.
[0413] In one embodiment, the second Fab molecule under b)
comprises a heavy chain variable region that is at least 95%, 96%,
97%, 98%, or 99% identical to the sequence of SEQ ID NO: 38, and a
light chain variable region that is at least 95%, 96%, 97%, 98%, or
99% identical to the sequence of SEQ ID NO: 39.
[0414] In still a further embodiment, the second Fab molecule under
b) comprises the heavy chain variable region sequence of SEQ ID NO:
38, and the light chain variable region sequence of SEQ ID NO:
39.
[0415] In a particular embodiment, the bispecific antibody
comprises a polypeptide that is at least 95%, 96%, 97%, 98%, or 99%
identical to the sequence of SEQ ID NO: 44, a polypeptide that is
at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of
SEQ ID NO: 45, a polypeptide that is at least 95%, 96%, 97%, 98%,
or 99% identical to the sequence of SEQ ID NO: 46, and a
polypeptide that is at least 95%, 96%, 97%, 98%, or 99% identical
to the sequence of SEQ ID NO: 47. In a further particular
embodiment, the bispecific antibody comprises a polypeptide
sequence of SEQ ID NO: 44, a polypeptide sequence of SEQ ID NO: 45,
a polypeptide sequence of SEQ ID NO: 46 and a polypeptide sequence
of SEQ ID NO: 47. (CD20XCD3 bsAB)
[0416] In one embodiment, the therapeutic agent comprises a
bispecific antibody comprising
[0417] (i) an antigen binding moiety that specifically binds to CD3
and comprises a heavy chain variable region comprising the heavy
chain HVR 1 (H1-HVR) of SEQ ID NO: 120, the H2-HVR of SEQ ID NO:
121, and the H3-HVR of SEQ ID NO: 122; and a light chain variable
region comprising the light chain HVR 1 (L1-HVR) of SEQ ID NO: 123,
the L2-HVR of SEQ ID NO: 124 and the L3-HVR of SEQ ID NO: 125;
and
[0418] (ii) an antigen binding moiety that specifically binds to
CD20 and comprises a heavy chain variable region comprising the
heavy chain HVR 1 (H1-HVR) of SEQ ID NO: 128, the H2-HVR of SEQ ID
NO: 129, and the H3-HVR of SEQ ID NO: 130; and a light chain
variable region comprising the light chain HVR 1 (L1-HVR) of SEQ ID
NO: 131, the L2-HVR of SEQ ID NO: 132 and the L3-HVR of SEQ ID NO:
133.
[0419] In one embodiment, the therapeutic agent comprises a
bispecific antibody comprising
[0420] (i) an antigen binding moiety that specifically binds to CD3
and comprises a heavy chain variable region of SEQ ID NO: 126; and
a light chain variable region of SEQ ID NO: 127; and
[0421] (ii) an antigen binding moiety that specifically binds to
CD20 and comprises a heavy chain variable region of SEQ ID NO: 134;
and a light chain variable region of SEQ ID NO: 135.
[0422] In one embodiment, the bispecific antibody comprises an
antigen binding moiety that specifically binds to CD19, and an
antigen binding moiety that specifically binds to CD3. In one
embodiment, the bispecific antibody comprises a first antigen
binding moiety that specifically binds to CD3, and a second and a
third antigen binding moiety that specifically bind to CD19. In one
embodiment, the first antigen binding moiety is a crossover Fab
molecule, and the second and the first antigen binding moiety are
each a conventional Fab molecule. In one embodiment, the bispecific
antibody further comprises an Fc domain. The bispecific antibody
may comprise modifications in the Fc region and/or the antigen
binding moieties as described herein.
[0423] In one embodiment, the therapeutic agent comprises a
bispecific antibody comprising
[0424] (i) an antigen binding moiety that specifically binds to CD3
and comprises a heavy chain variable region comprising the heavy
chain CDR (HCDR) 1 of SEQ ID NO: 32, the HCDR2 of SEQ ID NO: 33,
and the HCDR3 of SEQ ID NO: 34; and a light chain variable region
comprising the light chain CDR (LCDR) 1 of SEQ ID NO: 35, the LCDR2
of SEQ ID NO: 36 and the LCDR3 of SEQ ID NO: 37; and
[0425] (ii) an antigen binding moiety that specifically binds to
CD19 and comprises a heavy chain variable region comprising the
heavy chain CDR (HCDR) 1 of SEQ ID NO: 48, the HCDR2 of SEQ ID NO:
49, and the HCDR3 of SEQ ID NO: 50; and a light chain variable
region comprising the light chain CDR (LCDR) 1 of SEQ ID NO: 51,
the LCDR2 of SEQ ID NO: 52 and the LCDR3 of SEQ ID NO: 53.
[0426] In one embodiment, the therapeutic agent comprises a
bispecific antibody comprising
[0427] (i) an antigen binding moiety that specifically binds to CD3
and comprises a heavy chain variable region of SEQ ID NO: 38; and a
light chain variable region of SEQ ID NO: 39; and
[0428] (ii) an antigen binding moiety that specifically binds to
CD19 and comprises a heavy chain variable region of SEQ ID NO: 54;
and a light chain variable region of SEQ ID NO: 55.
[0429] In a particular embodiment, the therapeutic agent comprises
a bispecific antibody comprising
[0430] a) a first Fab molecule which specifically binds to a first
antigen;
[0431] b) a second Fab molecule which specifically binds to a
second antigen, and wherein the variable domains VL and VH of the
Fab light chain and the Fab heavy chain are replaced by each
other;
[0432] c) a third Fab molecule which specifically binds to the
first antigen; and
[0433] d) an Fc domain composed of a first and a second subunit
capable of stable association;
[0434] wherein
[0435] (i) the first antigen is CD19 and the second antigen is CD3,
particularly CD3 epsilon;
[0436] (ii) the first Fab molecule under a) and the third Fab
molecule under c) each comprise the heavy chain complementarity
determining region (CDR) 1 of SEQ ID NO: 48, the heavy chain CDR 2
of SEQ ID NO: 49, the heavy chain CDR 3 of SEQ ID NO: 50, the light
chain CDR 1 of SEQ ID NO: 51, the light chain CDR 2 of SEQ ID NO:
52 and the light chain CDR 3 of SEQ ID NO: 53, and the second Fab
molecule under b) comprises the heavy chain CDR 1 of SEQ ID NO: 32,
the heavy chain CDR 2 of SEQ ID NO: 33, the heavy chain CDR 3 of
SEQ ID NO: 34, the light chain CDR 1 of SEQ ID NO: 35, the light
chain CDR 2 of SEQ ID NO: 36 and the light chain CDR 3 of SEQ ID
NO: 37;
[0437] (iii) in the constant domain CL of the first Fab molecule
under a) and the third Fab molecule under c) the amino acid at
position 124 is substituted by lysine (K) (numbering according to
Kabat) and the amino acid at position 123 is substituted by lysine
(K) or arginine (R), particularly by arginine (R) (numbering
according to Kabat), and wherein in the constant domain CH1 of the
first Fab molecule under a) and the third Fab molecule under c) the
amino acid at position 147 is substituted by glutamic acid (E)
(numbering according to Kabat EU index) and the amino acid at
position 213 is substituted by glutamic acid (E) (numbering
according to Kabat EU index); and
[0438] (iv) the first Fab molecule under a) is fused at the
C-terminus of the Fab heavy chain to the N-terminus of the Fab
heavy chain of the second Fab molecule under b), and the second Fab
molecule under b) and the third Fab molecule under c) are each
fused at the C-terminus of the Fab heavy chain to the N-terminus of
one of the subunits of the Fc domain under d).
[0439] In one embodiment, the first Fab molecule under a) and the
third Fab molecule under c) each comprise a heavy chain variable
region that is at least 95%, 96%, 97%, 98%, or 99% identical to the
sequence of SEQ ID NO: 54, and a light chain variable region that
is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of
SEQ ID NO: 55.
[0440] In one embodiment, the first Fab molecule under a) and the
third Fab molecule under c) each comprise the heavy chain variable
region sequence of SEQ ID NO: 54, and the light chain variable
region sequence of SEQ ID NO: 55.
[0441] In one embodiment, the second Fab molecule under b)
comprises a heavy chain variable region that is at least 95%, 96%,
97%, 98%, or 99% identical to the sequence of SEQ ID NO: 38, and a
light chain variable region that is at least 95%, 96%, 97%, 98%, or
99% identical to the sequence of SEQ ID NO: 39.
[0442] In still a further embodiment, the second Fab molecule under
b) comprises the heavy chain variable region sequence of SEQ ID NO:
38, and the light chain variable region sequence of SEQ ID NO:
39.
[0443] In a particular embodiment, the bispecific antibody
comprises a polypeptide that is at least 95%, 96%, 97%, 98%, or 99%
identical to the sequence of SEQ ID NO: 47, a polypeptide that is
at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of
SEQ ID NO: 56, a polypeptide that is at least 95%, 96%, 97%, 98%,
or 99% identical to the sequence of SEQ ID NO: 57, and a
polypeptide that is at least 95%, 96%, 97%, 98%, or 99% identical
to the sequence of SEQ ID NO: 58. In a further particular
embodiment, the bispecific antibody comprises a polypeptide
sequence of SEQ ID NO: 47, a polypeptide sequence of SEQ ID NO: 56,
a polypeptide sequence of SEQ ID NO: 57 and a polypeptide sequence
of SEQ ID NO: 58.
[0444] In one embodiment, the therapeutic agent comprises a
bispecific antibody comprising
[0445] (i) an antigen binding moiety that specifically binds to CD3
and comprises a heavy chain variable region comprising the heavy
chain CDR (HCDR) 1 of SEQ ID NO: 32, the HCDR2 of SEQ ID NO: 33,
and the HCDR3 of SEQ ID NO: 34; and a light chain variable region
comprising the light chain CDR (LCDR) 1 of SEQ ID NO: 35, the LCDR2
of SEQ ID NO: 36 and the LCDR3 of SEQ ID NO: 37; and
[0446] (ii) an antigen binding moiety that specifically binds to
CD19 and comprises a heavy chain variable region comprising the
heavy chain CDR (HCDR) 1 of SEQ ID NO: 59, the HCDR2 of SEQ ID NO:
60, and the HCDR3 of SEQ ID NO: 61; and a light chain variable
region comprising the light chain CDR (LCDR) 1 of SEQ ID NO: 62,
the LCDR2 of SEQ ID NO: 63 and the LCDR3 of SEQ ID NO: 64.
[0447] In one embodiment, the therapeutic agent comprises a
bispecific antibody comprising
[0448] (i) an antigen binding moiety that specifically binds to CD3
and comprises a heavy chain variable region of SEQ ID NO: 38; and a
light chain variable region of SEQ ID NO: 39; and
[0449] (ii) an antigen binding moiety that specifically binds to
CD19 and comprises a heavy chain variable region of SEQ ID NO: 65;
and a light chain variable region of SEQ ID NO: 66.
[0450] In a particular embodiment, the therapeutic agent comprises
a bispecific antibody comprising
[0451] a) a first Fab molecule which specifically binds to a first
antigen;
[0452] b) a second Fab molecule which specifically binds to a
second antigen, and wherein the variable domains VL and VH of the
Fab light chain and the Fab heavy chain are replaced by each
other;
[0453] c) a third Fab molecule which specifically binds to the
first antigen; and
[0454] d) an Fc domain composed of a first and a second subunit
capable of stable association;
[0455] wherein
[0456] (i) the first antigen is CD19 and the second antigen is CD3,
particularly CD3 epsilon;
[0457] (ii) the first Fab molecule under a) and the third Fab
molecule under c) each comprise the heavy chain complementarity
determining region (CDR) 1 of SEQ ID NO: 59, the heavy chain CDR 2
of SEQ ID NO: 60, the heavy chain CDR 3 of SEQ ID NO: 61, the light
chain CDR 1 of SEQ ID NO: 62, the light chain CDR 2 of SEQ ID NO:
63 and the light chain CDR 3 of SEQ ID NO: 64, and the second Fab
molecule under b) comprises the heavy chain CDR 1 of SEQ ID NO: 32,
the heavy chain CDR 2 of SEQ ID NO: 33, the heavy chain CDR 3 of
SEQ ID NO: 34, the light chain CDR 1 of SEQ ID NO: 35, the light
chain CDR 2 of SEQ ID NO: 36 and the light chain CDR 3 of SEQ ID
NO: 37;
[0458] (iii) in the constant domain CL of the first Fab molecule
under a) and the third Fab molecule under c) the amino acid at
position 124 is substituted by lysine (K) (numbering according to
Kabat) and the amino acid at position 123 is substituted by lysine
(K) or arginine (R), particularly by arginine (R) (numbering
according to Kabat), and wherein in the constant domain CH1 of the
first Fab molecule under a) and the third Fab molecule under c) the
amino acid at position 147 is substituted by glutamic acid (E)
(numbering according to Kabat EU index) and the amino acid at
position 213 is substituted by glutamic acid (E) (numbering
according to Kabat EU index); and
[0459] (iv) the first Fab molecule under a) is fused at the
C-terminus of the Fab heavy chain to the N-terminus of the Fab
heavy chain of the second Fab molecule under b), and the second Fab
molecule under b) and the third Fab molecule under c) are each
fused at the C-terminus of the Fab heavy chain to the N-terminus of
one of the subunits of the Fc domain under d).
[0460] In one embodiment, the first Fab molecule under a) and the
third Fab molecule under c) each comprise a heavy chain variable
region that is at least 95%, 96%, 97%, 98%, or 99% identical to the
sequence of SEQ ID NO: 65, and a light chain variable region that
is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of
SEQ ID NO: 66.
[0461] In one embodiment, the first Fab molecule under a) and the
third Fab molecule under c) each comprise the heavy chain variable
region sequence of SEQ ID NO: 65, and the light chain variable
region sequence of SEQ ID NO: 66.
[0462] In one embodiment, the second Fab molecule under b)
comprises a heavy chain variable region that is at least 95%, 96%,
97%, 98%, or 99% identical to the sequence of SEQ ID NO: 38, and a
light chain variable region that is at least 95%, 96%, 97%, 98%, or
99% identical to the sequence of SEQ ID NO: 39.
[0463] In still a further embodiment, the second Fab molecule under
b) comprises the heavy chain variable region sequence of SEQ ID NO:
38, and the light chain variable region sequence of SEQ ID NO:
39.
[0464] In a particular embodiment, the bispecific antibody
comprises a polypeptide that is at least 95%, 96%, 97%, 98%, or 99%
identical to the sequence of SEQ ID NO: 47, a polypeptide that is
at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of
SEQ ID NO: 148, a polypeptide that is at least 95%, 96%, 97%, 98%,
or 99% identical to the sequence of SEQ ID NO: 149, and a
polypeptide that is at least 95%, 96%, 97%, 98%, or 99% identical
to the sequence of SEQ ID NO: 150. In a further particular
embodiment, the bispecific antibody comprises a polypeptide
sequence of SEQ ID NO: 47, a polypeptide sequence of SEQ ID NO:
148, a polypeptide sequence of SEQ ID NO: 149 and a polypeptide
sequence of SEQ ID NO: 150.
[0465] Antibody Formats
[0466] The components of an antibody comprised in the therapeutic
agent, particularly a multispecific antibody, can be fused to each
other in a variety of configurations. Exemplary configurations are
depicted in FIG. 6.
[0467] In particular embodiments, the antigen binding moieties
comprised in the antibody are Fab molecules. In such embodiments,
the first, second, third etc. antigen binding moiety may be
referred to herein as first, second, third etc. Fab molecule,
respectively. Furthermore, in particular embodiments, the antibody
comprises an Fc domain composed of a first and a second subunit
capable of stable association.
[0468] In some embodiments, the second Fab molecule is fused at the
C-terminus of the Fab heavy chain to the N-terminus of the first or
the second subunit of the Fc domain.
[0469] In one such embodiment, the first Fab molecule is fused at
the C-terminus of the Fab heavy chain to the N-terminus of the Fab
heavy chain of the second Fab molecule. In a specific such
embodiment, the antibody essentially consists of the first and the
second Fab molecule, the Fc domain composed of a first and a second
subunit, and optionally one or more peptide linkers, wherein the
first Fab molecule is fused at the C-terminus of the Fab heavy
chain to the N-terminus of the Fab heavy chain of the second Fab
molecule, and the second Fab molecule is fused at the C-terminus of
the Fab heavy chain to the N-terminus of the first or the second
subunit of the Fc domain. Such a configuration is schematically
depicted in FIGS. 6G and 6K. Optionally, the Fab light chain of the
first Fab molecule and the Fab light chain of the second Fab
molecule may additionally be fused to each other.
[0470] In another such embodiment, the first Fab molecule is fused
at the C-terminus of the Fab heavy chain to the N-terminus of the
first or second subunit of the Fc domain. In a specific such
embodiment, the antibody essentially consists of the first and the
second Fab molecule, the Fc domain composed of a first and a second
subunit, and optionally one or more peptide linkers, wherein the
first and the second Fab molecule are each fused at the C-terminus
of the Fab heavy chain to the N-terminus of one of the subunits of
the Fc domain. Such a configuration is schematically depicted in
FIGS. 6A and 6D. The first and the second Fab molecule may be fused
to the Fc domain directly or through a peptide linker. In a
particular embodiment the first and the second Fab molecule are
each fused to the Fc domain through an immunoglobulin hinge region.
In a specific embodiment, the immunoglobulin hinge region is a
human IgG.sub.1 hinge region, particularly where the Fc domain is
an IgG.sub.1 Fc domain.
[0471] In other embodiments, the first Fab molecule is fused at the
C-terminus of the Fab heavy chain to the N-terminus of the first or
second subunit of the Fc domain.
[0472] In one such embodiment, the second Fab molecule is fused at
the C-terminus of the Fab heavy chain to the N-terminus of the Fab
heavy chain of the first Fab molecule. In a specific such
embodiment, the antibody essentially consists of the first and the
second Fab molecule, the Fc domain composed of a first and a second
subunit, and optionally one or more peptide linkers, wherein the
second Fab molecule is fused at the C-terminus of the Fab heavy
chain to the N-terminus of the Fab heavy chain of the first Fab
molecule, and the first Fab molecule is fused at the C-terminus of
the Fab heavy chain to the N-terminus of the first or the second
subunit of the Fc domain. Such a configuration is schematically
depicted in FIGS. 6H and 6L. Optionally, the Fab light chain of the
first Fab molecule and the Fab light chain of the second Fab
molecule may additionally be fused to each other.
[0473] The Fab molecules may be fused to the Fc domain or to each
other directly or through a peptide linker, comprising one or more
amino acids, typically about 2-20 amino acids. Peptide linkers are
known in the art and are described herein. Suitable,
non-immunogenic peptide linkers include, for example,
(G.sub.4S).sub.n, (SG.sub.4).sub.n, (G.sub.4S).sub.n or
G.sub.4(SG.sub.4).sub.n peptide linkers. "n" is generally an
integer from 1 to 10, typically from 2 to 4. In one embodiment said
peptide linker has a length of at least 5 amino acids, in one
embodiment a length of 5 to 100, in a further embodiment of 10 to
50 amino acids. In one embodiment said peptide linker is
(GxS).sub.n or (GxS).sub.nG.sub.m with G=glycine, S=serine, and
(x=3, n=3, 4, 5 or 6, and m=0, 1, 2 or 3) or (x=4, n=2, 3, 4 or 5
and m=0, 1, 2 or 3), in one embodiment x=4 and n=2 or 3, in a
further embodiment x=4 and n=2. In one embodiment said peptide
linker is (G.sub.4S).sub.2. A particularly suitable peptide linker
for fusing the Fab light chains of the first and the second Fab
molecule to each other is (G.sub.4S).sub.2. An exemplary peptide
linker suitable for connecting the Fab heavy chains of the first
and the second Fab fragments comprises the sequence
(D)-(G.sub.4S).sub.2 (SEQ ID NOs 118 and 119). Another suitable
such linker comprises the sequence (G.sub.4S).sub.4. Additionally,
linkers may comprise (a portion of) an immunoglobulin hinge region.
Particularly where a Fab molecule is fused to the N-terminus of an
Fc domain subunit, it may be fused via an immunoglobulin hinge
region or a portion thereof, with or without an additional peptide
linker.
[0474] An antibody with a single antigen binding moiety (such as a
Fab molecule) capable of specific binding to a target cell antigen
(for example as shown in FIG. 6A, D, G, H, K, L) is useful,
particularly in cases where internalization of the target cell
antigen is to be expected following binding of a high affinity
antigen binding moiety. In such cases, the presence of more than
one antigen binding moiety specific for the target cell antigen may
enhance internalization of the target cell antigen, thereby
reducing its availability.
[0475] In many other cases, however, it will be advantageous to
have an antibody comprising two or more antigen binding moieties
(such as Fab molecules) specific for a target cell antigen (see
examples shown in FIG. 6B, 6C, 6E, 6F, 6I, 6J, 6M or 6N), for
example to optimize targeting to the target site or to allow
crosslinking of target cell antigens.
[0476] Accordingly, in particular embodiments, the antibody further
comprises a third Fab molecule which specifically binds to the
first antigen. The first antigen preferably is the target cell
antigen. In one embodiment, the third Fab molecule is a
conventional Fab molecule. In one embodiment, the third Fab
molecule is identical to the first Fab molecule (i.e. the first and
the third Fab molecule comprise the same heavy and light chain
amino acid sequences and have the same arrangement of domains (i.e.
conventional or crossover)). In a particular embodiment, the second
Fab molecule specifically binds to an activating T cell antigen,
particularly CD3, and the first and third Fab molecule specifically
bind to a target cell antigen.
[0477] In alternative embodiments, the antibody further comprises a
third Fab molecule which specifically binds to the second antigen.
In these embodiments, the second antigen preferably is the target
cell antigen. In one such embodiment, the third Fab molecule is a
crossover Fab molecule (a Fab molecule wherein the variable domains
VH and VL or the constant domains CL and CH1 of the Fab heavy and
light chains are exchanged/replaced by each other). In one such
embodiment, the third Fab molecule is identical to the second Fab
molecule (i.e. the second and the third Fab molecule comprise the
same heavy and light chain amino acid sequences and have the same
arrangement of domains (i.e. conventional or crossover)). In one
such embodiment, the first Fab molecule specifically binds to an
activating T cell antigen, particularly CD3, and the second and
third Fab molecule specifically bind to a target cell antigen.
[0478] In one embodiment, the third Fab molecule is fused at the
C-terminus of the Fab heavy chain to the N-terminus of the first or
second subunit of the Fc domain.
[0479] In a particular embodiment, the second and the third Fab
molecule are each fused at the C-terminus of the Fab heavy chain to
the N-terminus of one of the subunits of the Fc domain, and the
first Fab molecule is fused at the C-terminus of the Fab heavy
chain to the N-terminus of the Fab heavy chain of the second Fab
molecule. In a specific such embodiment, the antibody essentially
consists of the first, the second and the third Fab molecule, the
Fc domain composed of a first and a second subunit, and optionally
one or more peptide linkers, wherein the first Fab molecule is
fused at the C-terminus of the Fab heavy chain to the N-terminus of
the Fab heavy chain of the second Fab molecule, and the second Fab
molecule is fused at the C-terminus of the Fab heavy chain to the
N-terminus of the first subunit of the Fc domain, and wherein the
third Fab molecule is fused at the C-terminus of the Fab heavy
chain to the N-terminus of the second subunit of the Fc domain.
Such a configuration is schematically depicted in FIGS. 6B and 6E
(particular embodiments, wherein the third Fab molecule is a
conventional Fab molecule and preferably identical to the first Fab
molecule), and FIGS. 6I and 6M (alternative embodiments, wherein
the third Fab molecule is a crossover Fab molecule and preferably
identical to the second Fab molecule). The second and the third Fab
molecule may be fused to the Fc domain directly or through a
peptide linker. In a particular embodiment the second and the third
Fab molecule are each fused to the Fc domain through an
immunoglobulin hinge region. In a specific embodiment, the
immunoglobulin hinge region is a human IgG.sub.1 hinge region,
particularly where the Fc domain is an IgG.sub.1 Fc domain.
Optionally, the Fab light chain of the first Fab molecule and the
Fab light chain of the second Fab molecule may additionally be
fused to each other.
[0480] In another embodiment, the first and the third Fab molecule
are each fused at the C-terminus of the Fab heavy chain to the
N-terminus of one of the subunits of the Fc domain, and the second
Fab molecule is fused at the C-terminus of the Fab heavy chain to
the N-terminus of the Fab heavy chain of the first Fab molecule. In
a specific such embodiment, the antibody essentially consists of
the first, the second and the third Fab molecule, the Fc domain
composed of a first and a second subunit, and optionally one or
more peptide linkers, wherein the second Fab molecule is fused at
the C-terminus of the Fab heavy chain to the N-terminus of the Fab
heavy chain of the first Fab molecule, and the first Fab molecule
is fused at the C-terminus of the Fab heavy chain to the N-terminus
of the first subunit of the Fc domain, and wherein the third Fab
molecule is fused at the C-terminus of the Fab heavy chain to the
N-terminus of the second subunit of the Fc domain. Such a
configuration is schematically depicted in FIGS. 6C and 6F
(particular embodiments, wherein the third Fab molecule is a
conventional Fab molecule and preferably identical to the first Fab
molecule) and in FIGS. 6J and 6N (alternative embodiments, wherein
the third Fab molecule is a crossover Fab molecule and preferably
identical to the second Fab molecule). The first and the third Fab
molecule may be fused to the Fc domain directly or through a
peptide linker. In a particular embodiment the first and the third
Fab molecule are each fused to the Fc domain through an
immunoglobulin hinge region. In a specific embodiment, the
immunoglobulin hinge region is a human IgG.sub.1 hinge region,
particularly where the Fc domain is an IgG.sub.1 Fc domain.
Optionally, the Fab light chain of the first Fab molecule and the
Fab light chain of the second Fab molecule may additionally be
fused to each other.
[0481] In configurations of the antibody wherein a Fab molecule is
fused at the C-terminus of the Fab heavy chain to the N-terminus of
each of the subunits of the Fc domain through an immunoglobulin
hinge regions, the two Fab molecules, the hinge regions and the Fc
domain essentially form an immunoglobulin molecule. In a particular
embodiment the immunoglobulin molecule is an IgG class
immunoglobulin. In an even more particular embodiment the
immunoglobulin is an IgG.sub.1 subclass immunoglobulin. In another
embodiment the immunoglobulin is an IgG.sub.4 subclass
immunoglobulin. In a further particular embodiment the
immunoglobulin is a human immunoglobulin. In other embodiments the
immunoglobulin is a chimeric immunoglobulin or a humanized
immunoglobulin.
[0482] In some of the antibodies, the Fab light chain of the first
Fab molecule and the Fab light chain of the second Fab molecule are
fused to each other, optionally via a peptide linker. Depending on
the configuration of the first and the second Fab molecule, the Fab
light chain of the first Fab molecule may be fused at its
C-terminus to the N-terminus of the Fab light chain of the second
Fab molecule, or the Fab light chain of the second Fab molecule may
be fused at its C-terminus to the N-terminus of the Fab light chain
of the first Fab molecule. Fusion of the Fab light chains of the
first and the second Fab molecule further reduces mispairing of
unmatched Fab heavy and light chains, and also reduces the number
of plasmids needed for expression of some of the antibodies.
[0483] In certain embodiments the antibody comprises a polypeptide
wherein the Fab light chain variable region of the second Fab
molecule shares a carboxy-terminal peptide bond with the Fab heavy
chain constant region of the second Fab molecule (i.e. the second
Fab molecule comprises a crossover Fab heavy chain, wherein the
heavy chain variable region is replaced by a light chain variable
region), which in turn shares a carboxy-terminal peptide bond with
an Fc domain subunit (VL.sub.(2)-CH1.sub.(2)-CH2-CH3(-CH4)), and a
polypeptide wherein the Fab heavy chain of the first Fab molecule
shares a carboxy-terminal peptide bond with an Fc domain subunit
(VH.sub.(1)-CH1.sub.(1)-CH2-CH3(-CH4)). In some embodiments the
antibody further comprises a polypeptide wherein the Fab heavy
chain variable region of the second Fab molecule shares a
carboxy-terminal peptide bond with the Fab light chain constant
region of the second Fab molecule (VH.sub.(2)-CL.sub.(2)) and the
Fab light chain polypeptide of the first Fab molecule
(VL.sub.(1)-CL.sub.(1). In certain embodiments the polypeptides are
covalently linked, e.g., by a disulfide bond.
[0484] In certain embodiments the antibody comprises a polypeptide
wherein the Fab heavy chain variable region of the second Fab
molecule shares a carboxy-terminal peptide bond with the Fab light
chain constant region of the second Fab molecule (i.e. the second
Fab molecule comprises a crossover Fab heavy chain, wherein the
heavy chain constant region is replaced by a light chain constant
region), which in turn shares a carboxy-terminal peptide bond with
an Fc domain subunit (VH.sub.(2)-CL.sub.(2)-CH2-CH3(-CH4)), and a
polypeptide wherein the Fab heavy chain of the first Fab molecule
shares a carboxy-terminal peptide bond with an Fc domain subunit
(VH.sub.(1)-CH1.sub.(1)-CH2-CH3(-CH4)). In some embodiments the
antibody further comprises a polypeptide wherein the Fab light
chain variable region of the second Fab molecule shares a
carboxy-terminal peptide bond with the Fab heavy chain constant
region of the second Fab molecule (VL.sub.(2)-CH1.sub.(2)) and the
Fab light chain polypeptide of the first Fab molecule
(VL.sub.(1)-CL.sub.(1)). In certain embodiments the polypeptides
are covalently linked, e.g., by a disulfide bond.
[0485] In some embodiments, the antibody comprises a polypeptide
wherein the Fab light chain variable region of the second Fab
molecule shares a carboxy-terminal peptide bond with the Fab heavy
chain constant region of the second Fab molecule (i.e. the second
Fab molecule comprises a crossover Fab heavy chain, wherein the
heavy chain variable region is replaced by a light chain variable
region), which in turn shares a carboxy-terminal peptide bond with
the Fab heavy chain of the first Fab molecule, which in turn shares
a carboxy-terminal peptide bond with an Fc domain subunit
(VL.sub.(2)-CH1.sub.(2)-VH.sub.(1)-CH1.sub.(1)-CH2-CH3(-CH4)). In
other embodiments, the antibody comprises a polypeptide wherein the
Fab heavy chain of the first Fab molecule shares a carboxy-terminal
peptide bond with the Fab light chain variable region of the second
Fab molecule which in turn shares a carboxy-terminal peptide bond
with the Fab heavy chain constant region of the second Fab molecule
(i.e. the second Fab molecule comprises a crossover Fab heavy
chain, wherein the heavy chain variable region is replaced by a
light chain variable region), which in turn shares a
carboxy-terminal peptide bond with an Fc domain subunit
(VH.sub.(1)-CH1.sub.(1)-VL.sub.(2)-CH1.sub.(2)-CH2-CH3(-CH4)).
[0486] In some of these embodiments the antibody further comprises
a crossover Fab light chain polypeptide of the second Fab molecule,
wherein the Fab heavy chain variable region of the second Fab
molecule shares a carboxy-terminal peptide bond with the Fab light
chain constant region of the second Fab molecule
(VH.sub.(2)-CL.sub.(2)), and the Fab light chain polypeptide of the
first Fab molecule (VL.sub.(1)-CL.sub.(1)). In others of these
embodiments the antibody further comprises a polypeptide wherein
the Fab heavy chain variable region of the second Fab molecule
shares a carboxy-terminal peptide bond with the Fab light chain
constant region of the second Fab molecule which in turn shares a
carboxy-terminal peptide bond with the Fab light chain polypeptide
of the first Fab molecule
(VH.sub.(2)-CL.sub.(2)-VL.sub.(1)-CL.sub.(1)), or a polypeptide
wherein the Fab light chain polypeptide of the first Fab molecule
shares a carboxy-terminal peptide bond with the Fab heavy chain
variable region of the second Fab molecule which in turn shares a
carboxy-terminal peptide bond with the Fab light chain constant
region of the second Fab molecule
(VL.sub.(1)-CL.sub.(1)-VH.sub.(2)-CL.sub.(2)), as appropriate.
[0487] The antibody according to these embodiments may further
comprise (i) an Fc domain subunit polypeptide (CH2-CH3(-CH4)), or
(ii) a polypeptide wherein the Fab heavy chain of a third Fab
molecule shares a carboxy-terminal peptide bond with an Fc domain
subunit (VH.sub.(3)-CH1.sub.(3)-CH2-CH3(-CH4)) and the Fab light
chain polypeptide of a third Fab molecule (VL.sub.(3)-CL.sub.(3)).
In certain embodiments the polypeptides are covalently linked,
e.g., by a disulfide bond.
[0488] In some embodiments, the antibody comprises a polypeptide
wherein the Fab heavy chain variable region of the second Fab
molecule shares a carboxy-terminal peptide bond with the Fab light
chain constant region of the second Fab molecule (i.e. the second
Fab molecule comprises a crossover Fab heavy chain, wherein the
heavy chain constant region is replaced by a light chain constant
region), which in turn shares a carboxy-terminal peptide bond with
the Fab heavy chain of the first Fab molecule, which in turn shares
a carboxy-terminal peptide bond with an Fc domain subunit
(VH.sub.(2)-CL.sub.(2)-VH.sub.(1)-CH1.sub.(1)-CH2-CH3(-CH4)). In
other embodiments, the antibody comprises a polypeptide wherein the
Fab heavy chain of the first Fab molecule shares a carboxy-terminal
peptide bond with the Fab heavy chain variable region of the second
Fab molecule which in turn shares a carboxy-terminal peptide bond
with the Fab light chain constant region of the second Fab molecule
(i.e. the second Fab molecule comprises a crossover Fab heavy
chain, wherein the heavy chain constant region is replaced by a
light chain constant region), which in turn shares a
carboxy-terminal peptide bond with an Fc domain subunit
(VH.sub.(1)-CH1.sub.(1)-VH.sub.(2)-CL.sub.(2)-CH2-CH3(-CH4)).
[0489] In some of these embodiments the antibody further comprises
a crossover Fab light chain polypeptide of the second Fab molecule,
wherein the Fab light chain variable region of the second Fab
molecule shares a carboxy-terminal peptide bond with the Fab heavy
chain constant region of the second Fab molecule
(VL.sub.(2)-CH1.sub.(2)), and the Fab light chain polypeptide of
the first Fab molecule (VL.sub.(1)-CL.sub.(1)). In others of these
embodiments the antibody further comprises a polypeptide wherein
the Fab light chain variable region of the second Fab molecule
shares a carboxy-terminal peptide bond with the Fab heavy chain
constant region of the second Fab molecule which in turn shares a
carboxy-terminal peptide bond with the Fab light chain polypeptide
of the first Fab molecule
(VL.sub.(2)-CH1.sub.(2)-VL.sub.(1)-CL.sub.(1)), or a polypeptide
wherein the Fab light chain polypeptide of the first Fab molecule
shares a carboxy-terminal peptide bond with the Fab heavy chain
variable region of the second Fab molecule which in turn shares a
carboxy-terminal peptide bond with the Fab light chain constant
region of the second Fab molecule
(VL.sub.(1)-CL.sub.(1)-VH.sub.(2)-CL.sub.(2)), as appropriate.
[0490] The antibody according to these embodiments may further
comprise (i) an Fc domain subunit polypeptide (CH2-CH3(-CH4)), or
(ii) a polypeptide wherein the Fab heavy chain of a third Fab
molecule shares a carboxy-terminal peptide bond with an Fc domain
subunit (VH.sub.(3)-CH1.sub.(3)-CH2-CH3(-CH4)) and the Fab light
chain polypeptide of a third Fab molecule (VL.sub.(3)-CL.sub.(3)).
In certain embodiments the polypeptides are covalently linked,
e.g., by a disulfide bond.
[0491] In some embodiments, the first Fab molecule is fused at the
C-terminus of the Fab heavy chain to the N-terminus of the Fab
heavy chain of the second Fab molecule. In certain such
embodiments, the antibody does not comprise an Fc domain. In
certain embodiments, the antibody essentially consists of the first
and the second Fab molecule, and optionally one or more peptide
linkers, wherein the first Fab molecule is fused at the C-terminus
of the Fab heavy chain to the N-terminus of the Fab heavy chain of
the second Fab molecule. Such a configuration is schematically
depicted in FIGS. 6O and 6S.
[0492] In other embodiments, the second Fab molecule is fused at
the C-terminus of the Fab heavy chain to the N-terminus of the Fab
heavy chain of the first Fab molecule. In certain such embodiments,
the antibody does not comprise an Fc domain. In certain
embodiments, the antibody essentially consists of the first and the
second Fab molecule, and optionally one or more peptide linkers,
wherein the second Fab molecule is fused at the C-terminus of the
Fab heavy chain to the N-terminus of the Fab heavy chain of the
first Fab molecule. Such a configuration is schematically depicted
in FIGS. 6P and 6T.
[0493] In some embodiments, the first Fab molecule is fused at the
C-terminus of the Fab heavy chain to the N-terminus of the Fab
heavy chain of the second Fab molecule, and the antibody further
comprises a third Fab molecule, wherein said third Fab molecule is
fused at the C-terminus of the Fab heavy chain to the N-terminus of
the Fab heavy chain of the first Fab molecule. In particular such
embodiments, said third Fab molecule is a conventional Fab
molecule. In other such embodiments, said third Fab molecule is a
crossover Fab molecule as described herein, i.e. a Fab molecule
wherein the variable domains VH and VL or the constant domains CL
and CH1 of the Fab heavy and light chains are exchanged/replaced by
each other. In certain such embodiments, the antibody essentially
consists of the first, the second and the third Fab molecule, and
optionally one or more peptide linkers, wherein the first Fab
molecule is fused at the C-terminus of the Fab heavy chain to the
N-terminus of the Fab heavy chain of the second Fab molecule, and
the third Fab molecule is fused at the C-terminus of the Fab heavy
chain to the N-terminus of the Fab heavy chain of the first Fab
molecule. Such a configuration is schematically depicted in FIGS.
6Q and 6U (particular embodiments, wherein the third Fab molecule
is a conventional Fab molecule and preferably identical to the
first Fab molecule).
[0494] In some embodiments, the first Fab molecule is fused at the
C-terminus of the Fab heavy chain to the N-terminus of the Fab
heavy chain of the second Fab molecule, and the antibody further
comprises a third Fab molecule, wherein said third Fab molecule is
fused at the N-terminus of the Fab heavy chain to the C-terminus of
the Fab heavy chain of the second Fab molecule. In particular such
embodiments, said third Fab molecule is a crossover Fab molecule as
described herein, i.e. a Fab molecule wherein the variable domains
VH and VL or the constant domains CH1 and CL of the Fab heavy and
light chains are exchanged/replaced by each other. In other such
embodiments, said third Fab molecule is a conventional Fab
molecule. In certain such embodiments, the antibody essentially
consists of the first, the second and the third Fab molecule, and
optionally one or more peptide linkers, wherein the first Fab
molecule is fused at the C-terminus of the Fab heavy chain to the
N-terminus of the Fab heavy chain of the second Fab molecule, and
the third Fab molecule is fused at the N-terminus of the Fab heavy
chain to the C-terminus of the Fab heavy chain of the second Fab
molecule. Such a configuration is schematically depicted in FIGS.
6W and 6Y (particular embodiments, wherein the third Fab molecule
is a crossover Fab molecule and preferably identical to the second
Fab molecule).
[0495] In some embodiments, the second Fab molecule is fused at the
C-terminus of the Fab heavy chain to the N-terminus of the Fab
heavy chain of the first Fab molecule, and the antibody further
comprises a third Fab molecule, wherein said third Fab molecule is
fused at the N-terminus of the Fab heavy chain to the C-terminus of
the Fab heavy chain of the first Fab molecule. In particular such
embodiments, said third Fab molecule is a conventional Fab
molecule. In other such embodiments, said third Fab molecule is a
crossover Fab molecule as described herein, i.e. a Fab molecule
wherein the variable domains VH and VL or the constant domains CH1
and CL of the Fab heavy and light chains are exchanged/replaced by
each other. In certain such embodiments, the antibody essentially
consists of the first, the second and the third Fab molecule, and
optionally one or more peptide linkers, wherein the second Fab
molecule is fused at the C-terminus of the Fab heavy chain to the
N-terminus of the Fab heavy chain of the first Fab molecule, and
the third Fab molecule is fused at the N-terminus of the Fab heavy
chain to the C-terminus of the Fab heavy chain of the first Fab
molecule. Such a configuration is schematically depicted in FIGS.
6R and 6V (particular embodiments, wherein the third Fab molecule
is a conventional Fab molecule and preferably identical to the
first Fab molecule).
[0496] In some embodiments, the second Fab molecule is fused at the
C-terminus of the Fab heavy chain to the N-terminus of the Fab
heavy chain of the first Fab molecule, and the antibody further
comprises a third Fab molecule, wherein said third Fab molecule is
fused at the C-terminus of the Fab heavy chain to the N-terminus of
the Fab heavy chain of the second Fab molecule. In particular such
embodiments, said third Fab molecule is a crossover Fab molecule as
described herein, i.e. a Fab molecule wherein the variable domains
VH and VL or the constant domains CH1 and CL of the Fab heavy and
light chains are exchanged/replaced by each other. In other such
embodiments, said third Fab molecule is a conventional Fab
molecule. In certain such embodiments, the antibody essentially
consists of the first, the second and the third Fab molecule, and
optionally one or more peptide linkers, wherein the second Fab
molecule is fused at the C-terminus of the Fab heavy chain to the
N-terminus of the Fab heavy chain of the first Fab molecule, and
the third Fab molecule is fused at the C-terminus of the Fab heavy
chain to the N-terminus of the Fab heavy chain of the second Fab
molecule. Such a configuration is schematically depicted in FIGS.
6X and 6Z (particular embodiments, wherein the third Fab molecule
is a crossover Fab molecule and preferably identical to the first
Fab molecule).
[0497] In certain embodiments the antibody comprises a polypeptide
wherein the Fab heavy chain of the first Fab molecule shares a
carboxy-terminal peptide bond with the Fab light chain variable
region of the second Fab molecule, which in turn shares a
carboxy-terminal peptide bond with the Fab heavy chain constant
region of the second Fab molecule (i.e. the second Fab molecule
comprises a crossover Fab heavy chain, wherein the heavy chain
variable region is replaced by a light chain variable region)
(VH.sub.(1)-CH1.sub.(1)-VL.sub.(2)-CH1.sub.(2)). In some
embodiments the antibody further comprises a polypeptide wherein
the Fab heavy chain variable region of the second Fab molecule
shares a carboxy-terminal peptide bond with the Fab light chain
constant region of the second Fab molecule (VH.sub.(2)-CL.sub.(2))
and the Fab light chain polypeptide of the first Fab molecule
(VL.sub.(1)-CL.sub.(1)).
[0498] In certain embodiments the antibody comprises a polypeptide
wherein the Fab light chain variable region of the second Fab
molecule shares a carboxy-terminal peptide bond with the Fab heavy
chain constant region of the second Fab molecule (i.e. the second
Fab molecule comprises a crossover Fab heavy chain, wherein the
heavy chain variable region is replaced by a light chain variable
region), which in turn shares a carboxy-terminal peptide bond with
the Fab heavy chain of the first Fab molecule
(VL.sub.(2)-CH1.sub.(2)-VH.sub.(1)-CH1.sub.(1)). In some
embodiments the antibody further comprises a polypeptide wherein
the Fab heavy chain variable region of the second Fab molecule
shares a carboxy-terminal peptide bond with the Fab light chain
constant region of the second Fab molecule (VH.sub.(2)-CL.sub.(2))
and the Fab light chain polypeptide of the first Fab molecule
(VL.sub.(1)-CL.sub.(1)).
[0499] In certain embodiments the antibody comprises a polypeptide
wherein the Fab heavy chain variable region of the second Fab
molecule shares a carboxy-terminal peptide bond with the Fab light
chain constant region of the second Fab molecule (i.e. the second
Fab molecule comprises a crossover Fab heavy chain, wherein the
heavy chain constant region is replaced by a light chain constant
region), which in turn shares a carboxy-terminal peptide bond with
the Fab heavy chain of the first Fab molecule
(VH.sub.(2)-CL.sub.(2)-VH.sub.(1)-CH1.sub.(1)). In some embodiments
the antibody further comprises a polypeptide wherein the Fab light
chain variable region of the second Fab molecule shares a
carboxy-terminal peptide bond with the Fab heavy chain constant
region of the second Fab molecule (VL.sub.(2)-CH1.sub.(2)) and the
Fab light chain polypeptide of the first Fab molecule
(VL.sub.(1)-CL.sub.(1)).
[0500] In certain embodiments the antibody comprises a polypeptide
wherein the Fab heavy chain of a third Fab molecule shares a
carboxy-terminal peptide bond with the Fab heavy chain of the first
Fab molecule, which in turn shares a carboxy-terminal peptide bond
with the Fab light chain variable region of the second Fab
molecule, which in turn shares a carboxy-terminal peptide bond with
the Fab heavy chain constant region of the second Fab molecule
(i.e. the second Fab molecule comprises a crossover Fab heavy
chain, wherein the heavy chain variable region is replaced by a
light chain variable region)
(VH.sub.(3)-CH1.sub.(3)-VH.sub.(1)-CH1.sub.(1)-VL.sub.(2)-CH1.sub.(2)).
In some embodiments the antibody further comprises a polypeptide
wherein the Fab heavy chain variable region of the second Fab
molecule shares a carboxy-terminal peptide bond with the Fab light
chain constant region of the second Fab molecule
(VH.sub.(2)-CL.sub.(2)) and the Fab light chain polypeptide of the
first Fab molecule (VL.sub.(1)-CL.sub.(1)). In some embodiments the
antibody further comprises the Fab light chain polypeptide of a
third Fab molecule (VL.sub.(3)-CL.sub.(3)).
[0501] In certain embodiments the antibody comprises a polypeptide
wherein the Fab heavy chain of a third Fab molecule shares a
carboxy-terminal peptide bond with the Fab heavy chain of the first
Fab molecule, which in turn shares a carboxy-terminal peptide bond
with the Fab heavy chain variable region of the second Fab
molecule, which in turn shares a carboxy-terminal peptide bond with
the Fab light chain constant region of the second Fab molecule
(i.e. the second Fab molecule comprises a crossover Fab heavy
chain, wherein the heavy chain constant region is replaced by a
light chain constant region)
(VH.sub.(3)-CH1.sub.(3)-VH.sub.(1)-CH1.sub.(1)-VH.sub.(2)-CL.sub.(2)).
In some embodiments the antibody further comprises a polypeptide
wherein the Fab light chain variable region of the second Fab
molecule shares a carboxy-terminal peptide bond with the Fab heavy
chain constant region of the second Fab molecule
(VL.sub.(2)-CH1.sub.(2)) and the Fab light chain polypeptide of the
first Fab molecule (VL.sub.(1)-CL.sub.(1)). In some embodiments the
antibody further comprises the Fab light chain polypeptide of a
third Fab molecule (VL.sub.(3)-CL.sub.(3)).
[0502] In certain embodiments the antibody comprises a polypeptide
wherein the Fab light chain variable region of the second Fab
molecule shares a carboxy-terminal peptide bond with the Fab heavy
chain constant region of the second Fab molecule (i.e. the second
Fab molecule comprises a crossover Fab heavy chain, wherein the
heavy chain variable region is replaced by a light chain variable
region), which in turn shares a carboxy-terminal peptide bond with
the Fab heavy chain of the first Fab molecule, which in turn shares
a carboxy-terminal peptide bond with the Fab heavy chain of a third
Fab molecule
(VL.sub.(2)-CH1.sub.(2)-VH.sub.(1)-CH1.sub.(1)-VH.sub.(3)-CH1.sub.(3)).
In some embodiments the antibody further comprises a polypeptide
wherein the Fab heavy chain variable region of the second Fab
molecule shares a carboxy-terminal peptide bond with the Fab light
chain constant region of the second Fab molecule
(VH.sub.(2)-CL.sub.(2)) and the Fab light chain polypeptide of the
first Fab molecule (VL.sub.(1)-CL.sub.(1)). In some embodiments the
antibody further comprises the Fab light chain polypeptide of a
third Fab molecule (VL.sub.(3)-CL.sub.(3)).
[0503] In certain embodiments the antibody comprises a polypeptide
wherein the Fab heavy chain variable region of the second Fab
molecule shares a carboxy-terminal peptide bond with the Fab light
chain constant region of the second Fab molecule (i.e. the second
Fab molecule comprises a crossover Fab heavy chain, wherein the
heavy chain constant region is replaced by a light chain constant
region), which in turn shares a carboxy-terminal peptide bond with
the Fab heavy chain of the first Fab molecule, which in turn shares
a carboxy-terminal peptide bond with the Fab heavy chain of a third
Fab molecule
(VH.sub.(2)-CL.sub.(2)-VH.sub.(1)-CH1.sub.(1)-VH.sub.(3)-CH1.sub.(3)).
In some embodiments the antibody further comprises a polypeptide
wherein the Fab light chain variable region of the second Fab
molecule shares a carboxy-terminal peptide bond with the Fab heavy
chain constant region of the second Fab molecule
(VL.sub.(2)-CH1.sub.(2)) and the Fab light chain polypeptide of the
first Fab molecule (VL.sub.(1)-CL.sub.(1)). In some embodiments the
antibody further comprises the Fab light chain polypeptide of a
third Fab molecule (VL.sub.(3)-CL.sub.(3)).
[0504] In certain embodiments the antibody comprises a polypeptide
wherein the Fab heavy chain of the first Fab molecule shares a
carboxy-terminal peptide bond with the Fab light chain variable
region of the second Fab molecule, which in turn shares a
carboxy-terminal peptide bond with the Fab heavy chain constant
region of the second Fab molecule (i.e. the second Fab molecule
comprises a crossover Fab heavy chain, wherein the heavy chain
variable region is replaced by a light chain variable region),
which in turn shares a carboxy-terminal peptide bond with the Fab
light chain variable region of a third Fab molecule, which in turn
shares a carboxy-terminal peptide bond with the Fab heavy chain
constant region of a third Fab molecule (i.e. the third Fab
molecule comprises a crossover Fab heavy chain, wherein the heavy
chain variable region is replaced by a light chain variable region)
(VH.sub.(1)-CH1.sub.(1)-VL.sub.(2)-CH1.sub.(2)-VL.sub.(3)-CH1.sub.(3)).
In some embodiments the antibody further comprises a polypeptide
wherein the Fab heavy chain variable region of the second Fab
molecule shares a carboxy-terminal peptide bond with the Fab light
chain constant region of the second Fab molecule
(VH.sub.(2)-CL.sub.(2)) and the Fab light chain polypeptide of the
first Fab molecule (VL.sub.(1)-CL.sub.(1)). In some embodiments the
antibody further comprises a polypeptide wherein the Fab heavy
chain variable region of a third Fab molecule shares a
carboxy-terminal peptide bond with the Fab light chain constant
region of a third Fab molecule (VH.sub.(3)-CL.sub.(3)).
[0505] In certain embodiments the antibody comprises a polypeptide
wherein the Fab heavy chain of the first Fab molecule shares a
carboxy-terminal peptide bond with the Fab heavy chain variable
region of the second Fab molecule, which in turn shares a
carboxy-terminal peptide bond with the Fab light chain constant
region of the second Fab molecule (i.e. the second Fab molecule
comprises a crossover Fab heavy chain, wherein the heavy chain
constant region is replaced by a light chain constant region),
which in turn shares a carboxy-terminal peptide bond with the Fab
heavy chain variable region of a third Fab molecule, which in turn
shares a carboxy-terminal peptide bond with the Fab light chain
constant region of a third Fab molecule (i.e. the third Fab
molecule comprises a crossover Fab heavy chain, wherein the heavy
chain constant region is replaced by a light chain constant region)
(VH.sub.(1)-CH1.sub.(1)-VH.sub.(2)-CL.sub.(2)-VH.sub.(3)-CL.sub.(3)).
In some embodiments the antibody further comprises a polypeptide
wherein the Fab light chain variable region of the second Fab
molecule shares a carboxy-terminal peptide bond with the Fab heavy
chain constant region of the second Fab molecule
(VL.sub.(2)-CH1.sub.(2)) and the Fab light chain polypeptide of the
first Fab molecule (VL.sub.(1)-CL.sub.(1)). In some embodiments the
antibody further comprises a polypeptide wherein the Fab light
chain variable region of a third Fab molecule shares a
carboxy-terminal peptide bond with the Fab heavy chain constant
region of a third Fab molecule (VL.sub.(3)-CH1.sub.(3)).
[0506] In certain embodiments the antibody comprises a polypeptide
wherein the Fab light chain variable region of a third Fab molecule
shares a carboxy-terminal peptide bond with the Fab heavy chain
constant region of a third Fab molecule (i.e. the third Fab
molecule comprises a crossover Fab heavy chain, wherein the heavy
chain variable region is replaced by a light chain variable
region), which in turn shares a carboxy-terminal peptide bond with
the Fab light chain variable region of the second Fab molecule,
which in turn shares a carboxy-terminal peptide bond with the Fab
heavy chain constant region of the second Fab molecule (i.e. the
second Fab molecule comprises a crossover Fab heavy chain, wherein
the heavy chain variable region is replaced by a light chain
variable region), which in turn shares a carboxy-terminal peptide
bond with the Fab heavy chain of the first Fab molecule
(VL.sub.(3)-CH1.sub.(3)-VL.sub.(2)-CH1.sub.(2)-VH.sub.(1)-CH1.sub.(1)).
In some embodiments the antibody further comprises a polypeptide
wherein the Fab heavy chain variable region of the second Fab
molecule shares a carboxy-terminal peptide bond with the Fab light
chain constant region of the second Fab molecule
(VH.sub.(2)-CL.sub.(2)) and the Fab light chain polypeptide of the
first Fab molecule (VL.sub.(1)-CL.sub.(1)). In some embodiments the
antibody further comprises a polypeptide wherein the Fab heavy
chain variable region of a third Fab molecule shares a
carboxy-terminal peptide bond with the Fab light chain constant
region of a third Fab molecule (VH.sub.(3)-CL.sub.(3)).
[0507] In certain embodiments the antibody comprises a polypeptide
wherein the Fab heavy chain variable region of a third Fab molecule
shares a carboxy-terminal peptide bond with the Fab light chain
constant region of a third Fab molecule (i.e. the third Fab
molecule comprises a crossover Fab heavy chain, wherein the heavy
chain constant region is replaced by a light chain constant
region), which in turn shares a carboxy-terminal peptide bond with
the Fab heavy chain variable region of the second Fab molecule,
which in turn shares a carboxy-terminal peptide bond with the Fab
light chain constant region of the second Fab molecule (i.e. the
second Fab molecule comprises a crossover Fab heavy chain, wherein
the heavy chain constant region is replaced by a light chain
constant region), which in turn shares a carboxy-terminal peptide
bond with the Fab heavy chain of the first Fab molecule
(VH.sub.(3)-CL.sub.(3)-VH.sub.(2)-CL.sub.(2)-VH.sub.(1)-CH1.sub.(1)).
In some embodiments the antibody further comprises a polypeptide
wherein the Fab light chain variable region of the second Fab
molecule shares a carboxy-terminal peptide bond with the Fab heavy
chain constant region of the second Fab molecule
(VL.sub.(2)-CH1.sub.(2)) and the Fab light chain polypeptide of the
first Fab molecule (VL.sub.(1)-CL.sub.(1)). In some embodiments the
antibody further comprises a polypeptide wherein the Fab light
chain variable region of a third Fab molecule shares a
carboxy-terminal peptide bond with the Fab heavy chain constant
region of a third Fab molecule (VL.sub.(3)-CH1.sub.(3)).
[0508] According to any of the above embodiments, components of the
antibody (e.g. Fab molecules, Fc domain) may be fused directly or
through various linkers, particularly peptide linkers comprising
one or more amino acids, typically about 2-20 amino acids, that are
described herein or are known in the art. Suitable, non-immunogenic
peptide linkers include, for example, (G.sub.4S).sub.n,
(SG.sub.4).sub.n, (G.sub.4S).sub.n or G.sub.4(SG.sub.4).sub.n
peptide linkers, wherein n is generally an integer from 1 to 10,
typically from 2 to 4.
[0509] Fc Domain
[0510] An antibody, e.g. a bispecific antibody or an
immunoconjugate, comprised in the therapeutic agent may comprise an
Fc domain which consists of a pair of polypeptide chains comprising
heavy chain domains of an antibody molecule. For example, the Fc
domain of an immunoglobulin G (IgG) molecule is a dimer, each
subunit of which comprises the CH2 and CH3 IgG heavy chain constant
domains. The two subunits of the Fc domain are capable of stable
association with each other.
[0511] In one embodiment, the Fc domain is an IgG Fc domain. In a
particular embodiment the Fc domain is an IgG.sub.1 Fc domain. In
another embodiment the Fc domain is an IgG.sub.4 Fc domain. In a
more specific embodiment, the Fc domain is an IgG.sub.4 Fc domain
comprising an amino acid substitution at position S228 (Kabat
numbering), particularly the amino acid substitution S228P. This
amino acid substitution reduces in vivo Fab arm exchange of
IgG.sub.4 antibodies (see Stubenrauch et al., Drug Metabolism and
Disposition 38, 84-91 (2010)). In a further particular embodiment
the Fc domain is human. An exemplary sequence of a human IgG.sub.1
Fc region is given in SEQ ID NO: 30.
[0512] (i) Fc Domain Modifications Promoting Heterodimerization
[0513] Antibodies, particularly bispecific antibodies or
immunoconjugates, comprised in the therapeutic agent may comprise
different components (e.g. antigen binding domains, cytokines)
fused to one or the other of the two subunits of the Fc domain,
thus the two subunits of the Fc domain are typically comprised in
two non-identical polypeptide chains. Recombinant co-expression of
these polypeptides and subsequent dimerization leads to several
possible combinations of the two polypeptides. To improve the yield
and purity of such antibodies in recombinant production, it will
thus be advantageous to introduce in the Fc domain of the antibody
a modification promoting the association of the desired
polypeptides.
[0514] Accordingly, in particular embodiments the Fc domain
comprises a modification promoting the association of the first and
the second subunit of the Fc domain. The site of most extensive
protein-protein interaction between the two subunits of a human IgG
Fc domain is in the CH3 domain of the Fc domain. Thus, in one
embodiment said modification is in the CH3 domain of the Fc
domain.
[0515] There exist several approaches for modifications in the CH3
domain of the Fc domain in order to enforce heterodimerization,
which are well described e.g. in WO 96/27011, WO 98/050431, EP
1870459, WO 2007/110205, WO 2007/147901, WO 2009/089004, WO
2010/129304, WO 2011/90754, WO 2011/143545, WO 2012058768, WO
2013157954, WO 2013096291. Typically, in all such approaches the
CH3 domain of the first subunit of the Fc domain and the CH3 domain
of the second subunit of the Fc domain are both engineered in a
complementary manner so that each CH3 domain (or the heavy chain
comprising it) can no longer homodimerize with itself but is forced
to heterodimerize with the complementarily engineered other CH3
domain (so that the first and second CH3 domain heterodimerize and
no homodimers between the two first or the two second CH3 domains
are formed). These different approaches for improved heavy chain
heterodimerization are contemplated as different alternatives in
combination with heavy-light chain modifications (e.g. variable or
constant region exchange/replacement in Fab arms, or introduction
of substitutions of charged amino acids with opposite charges in
the CH1/CL interface) which reduce light chain mispairing and Bence
Jones-type side products.
[0516] In a specific embodiment said modification promoting the
association of the first and the second subunit of the Fc domain is
a so-called "knob-into-hole" modification, comprising a "knob"
modification in one of the two subunits of the Fc domain and a
"hole" modification in the other one of the two subunits of the Fc
domain.
[0517] The knob-into-hole technology is described e.g. in U.S. Pat.
Nos. 5,731,168; 7,695,936; Ridgway et al., Prot Eng 9, 617-621
(1996) and Carter, J Immunol Meth 248, 7-15 (2001). Generally, the
method involves introducing a protuberance ("knob") at the
interface of a first polypeptide and a corresponding cavity
("hole") in the interface of a second polypeptide, such that the
protuberance can be positioned in the cavity so as to promote
heterodimer formation and hinder homodimer formation. Protuberances
are constructed by replacing small amino acid side chains from the
interface of the first polypeptide with larger side chains (e.g.
tyrosine or tryptophan). Compensatory cavities of identical or
similar size to the protuberances are created in the interface of
the second polypeptide by replacing large amino acid side chains
with smaller ones (e.g. alanine or threonine).
[0518] Accordingly, in a particular embodiment, in the CH3 domain
of the first subunit of the Fc domain an amino acid residue is
replaced with an amino acid residue having a larger side chain
volume, thereby generating a protuberance within the CH3 domain of
the first subunit which is positionable in a cavity within the CH3
domain of the second subunit, and in the CH3 domain of the second
subunit of the Fc domain an amino acid residue is replaced with an
amino acid residue having a smaller side chain volume, thereby
generating a cavity within the CH3 domain of the second subunit
within which the protuberance within the CH3 domain of the first
subunit is positionable.
[0519] Preferably said amino acid residue having a larger side
chain volume is selected from the group consisting of arginine (R),
phenylalanine (F), tyrosine (Y), and tryptophan (W).
[0520] Preferably said amino acid residue having a smaller side
chain volume is selected from the group consisting of alanine (A),
serine (S), threonine (T), and valine (V).
[0521] The protuberance and cavity can be made by altering the
nucleic acid encoding the polypeptides, e.g. by site-specific
mutagenesis, or by peptide synthesis.
[0522] In a specific embodiment, in the CH3 domain of the first
subunit of the Fc domain (the "knobs" subunit) the threonine
residue at position 366 is replaced with a tryptophan residue
(T366W), and in the CH3 domain of the second subunit of the Fc
domain (the "hole" subunit) the tyrosine residue at position 407 is
replaced with a valine residue (Y407V). In one embodiment, in the
second subunit of the Fc domain additionally the threonine residue
at position 366 is replaced with a serine residue (T366S) and the
leucine residue at position 368 is replaced with an alanine residue
(L368A) (numberings according to Kabat EU index).
[0523] In yet a further embodiment, in the first subunit of the Fc
domain additionally the serine residue at position 354 is replaced
with a cysteine residue (S354C) or the glutamic acid residue at
position 356 is replaced with a cysteine residue (E356C), and in
the second subunit of the Fc domain additionally the tyrosine
residue at position 349 is replaced by a cysteine residue (Y349C)
(numberings according to Kabat EU index). Introduction of these two
cysteine residues results in formation of a disulfide bridge
between the two subunits of the Fc domain, further stabilizing the
dimer (Carter, J Immunol Methods 248, 7-15 (2001)).
[0524] In a particular embodiment, the first subunit of the Fc
domain comprises amino acid substitutions S354C and T366W, and the
second subunit of the Fc domain comprises amino acid substitutions
Y349C, T366S, L368A and Y407V (numbering according to Kabat EU
index).
[0525] In a particular embodiment the mutant IL-2 polypeptide in
the immunoconjugate described herein, or the CD3 antigen binding
moiety in the bispecific antibody described herein, is fused to the
first subunit of the Fc domain (comprising the "knob"
modification). Without wishing to be bound by theory, fusion of the
IL-2 polypeptide or CD3 antigen binding moiety to the
knob-containing subunit of the Fc domain will (further) minimize
the generation of immunoconjugates comprising two IL-2 polypeptides
or bispecific antibodies comprising two CD3 antigen binding
moieties, respectively (steric clash of two knob-containing
polypeptides).
[0526] Other techniques of CH3-modification for enforcing the
heterodimerization are contemplated as alternatives according to
the invention and are described e.g. in WO 96/27011, WO 98/050431,
EP 1870459, WO 2007/110205, WO 2007/147901, WO 2009/089004, WO
2010/129304, WO 2011/90754, WO 2011/143545, WO 2012/058768, WO
2013/157954, WO 2013/096291.
[0527] In one embodiment the heterodimerization approach described
in EP 1870459 A1, is used alternatively. This approach is based on
the introduction of charged amino acids with opposite charges at
specific amino acid positions in the CH3/CH3 domain interface
between the two subunits of the Fc domain. One preferred embodiment
are amino acid mutations R409D; K370E in one of the two CH3 domains
(of the Fc domain) and amino acid mutations D399K; E357K in the
other one of the CH3 domains of the Fc domain (numbering according
to Kabat EU index).
[0528] In another embodiment the antibody comprises amino acid
mutation T366W in the CH3 domain of the first subunit of the Fc
domain and amino acid mutations T366S, L368A, Y407V in the CH3
domain of the second subunit of the Fc domain, and additionally
amino acid mutations R409D; K370E in the CH3 domain of the first
subunit of the Fc domain and amino acid mutations D399K; E357K in
the CH3 domain of the second subunit of the Fc domain (numberings
according to Kabat EU index).
[0529] In another embodiment the antibody comprises amino acid
mutations S354C, T366W in the CH3 domain of the first subunit of
the Fc domain and amino acid mutations Y349C, T366S, L368A, Y407V
in the CH3 domain of the second subunit of the Fc domain, or the
antibody comprises amino acid mutations Y349C, T366W in the CH3
domain of the first subunit of the Fc domain and amino acid
mutations S354C, T366S, L368A, Y407V in the CH3 domains of the
second subunit of the Fc domain and additionally amino acid
mutations R409D; K370E in the CH3 domain of the first subunit of
the Fc domain and amino acid mutations D399K; E357K in the CH3
domain of the second subunit of the Fc domain (all numberings
according to Kabat EU index).
[0530] In one embodiment the heterodimerization approach described
in WO 2013/157953 is used alternatively. In one embodiment a first
CH3 domain comprises amino acid mutation T366K and a second CH3
domain comprises amino acid mutation L351D (numberings according to
Kabat EU index). In a further embodiment the first CH3 domain
comprises further amino acid mutation L351K. In a further
embodiment the second CH3 domain comprises further an amino acid
mutation selected from Y349E, Y349D and L368E (preferably L368E)
(numberings according to Kabat EU index).
[0531] In one embodiment the heterodimerization approach described
in WO 2012/058768 is used alternatively. In one embodiment a first
CH3 domain comprises amino acid mutations L351Y, Y407A and a second
CH3 domain comprises amino acid mutations T366A, K409F. In a
further embodiment the second CH3 domain comprises a further amino
acid mutation at position T411, D399, 5400, F405, N390, or K392,
e.g. selected from a) T411N, T411R, T411Q, T411K, T411D, T411E or
T411W, b) D399R, D399W, D399Y or D399K, c) S400E, S400D, S400R, or
S400K, d) F405I, F405M, F405T, F405S, F405V or F405W, e) N390R,
N390K or N390D, f) K392V, K392M, K392R, K392L, K392F or K392E
(numberings according to Kabat EU index). In a further embodiment a
first CH3 domain comprises amino acid mutations L351Y, Y407A and a
second CH3 domain comprises amino acid mutations T366V, K409F. In a
further embodiment a first CH3 domain comprises amino acid mutation
Y407A and a second CH3 domain comprises amino acid mutations T366A,
K409F. In a further embodiment the second CH3 domain further
comprises amino acid mutations K392E, T411E, D399R and S400R
(numberings according to Kabat EU index).
[0532] In one embodiment the heterodimerization approach described
in WO 2011/143545 is used alternatively, e.g. with the amino acid
modification at a position selected from the group consisting of
368 and 409 (numbering according to Kabat EU index).
[0533] In one embodiment the heterodimerization approach described
in WO 2011/090762, which also uses the knobs-into-holes technology
described above, is used alternatively. In one embodiment a first
CH3 domain comprises amino acid mutation T366W and a second CH3
domain comprises amino acid mutation Y407A. In one embodiment a
first CH3 domain comprises amino acid mutation T366Y and a second
CH3 domain comprises amino acid mutation Y407T (numberings
according to Kabat EU index).
[0534] In one embodiment the antibody or its Fc domain is of IgG2
subclass and the heterodimerization approach described in WO
2010/129304 is used alternatively.
[0535] In an alternative embodiment a modification promoting
association of the first and the second subunit of the Fc domain
comprises a modification mediating electrostatic steering effects,
e.g. as described in PCT publication WO 2009/089004. Generally,
this method involves replacement of one or more amino acid residues
at the interface of the two Fc domain subunits by charged amino
acid residues so that homodimer formation becomes electrostatically
unfavorable but heterodimerization electrostatically favorable. In
one such embodiment a first CH3 domain comprises amino acid
substitution of K392 or N392 with a negatively charged amino acid
(e.g. glutamic acid (E), or aspartic acid (D), preferably K392D or
N392D) and a second CH3 domain comprises amino acid substitution of
D399, E356, D356, or E357 with a positively charged amino acid
(e.g. lysine (K) or arginine (R), preferably D399K, E356K, D356K,
or E357K, and more preferably D399K and E356K). In a further
embodiment the first CH3 domain further comprises amino acid
substitution of K409 or R409 with a negatively charged amino acid
(e.g. glutamic acid (E), or aspartic acid (D), preferably K409D or
R409D). In a further embodiment the first CH3 domain further or
alternatively comprises amino acid substitution of K439 and/or K370
with a negatively charged amino acid (e.g. glutamic acid (E), or
aspartic acid (D)) (all numberings according to Kabat EU
index).
[0536] In yet a further embodiment the heterodimerization approach
described in WO 2007/147901 is used alternatively. In one
embodiment a first CH3 domain comprises amino acid mutations K253E,
D282K, and K322D and a second CH3 domain comprises amino acid
mutations D239K, E240K, and K292D (numberings according to Kabat EU
index).
[0537] In still another embodiment the heterodimerization approach
described in WO 2007/110205 can be used alternatively.
[0538] In one embodiment, the first subunit of the Fc domain
comprises amino acid substitutions K392D and K409D, and the second
subunit of the Fc domain comprises amino acid substitutions D356K
and D399K (numbering according to Kabat EU index).
[0539] (ii) Fc Domain Modifications Reducing Fc Receptor Binding
and/or Effector Function
[0540] The Fc domain confers to an antibody, such as a bispecific
antibody or immunoconjugate, favorable pharmacokinetic properties,
including a long serum half-life which contributes to good
accumulation in the target tissue and a favorable tissue-blood
distribution ratio. At the same time it may, however, lead to
undesirable targeting of the antibody to cells expressing Fc
receptors rather than to the preferred antigen-bearing cells.
Moreover, the co-activation of Fc receptor signaling pathways may
lead to cytokine release which, in combination with other
immunostimulatory properties the antibody may have and the long
half-life of the antibody, results in excessive activation of
cytokine receptors and severe side effects upon systemic
administration.
[0541] Accordingly, in particular embodiments, the Fc domain of the
antibody, particularly bispecific antibody or immunoconjugate,
comprised in the therapeutic agent exhibits reduced binding
affinity to an Fc receptor and/or reduced effector function, as
compared to a native IgG.sub.1 Fc domain. In one such embodiment
the Fc domain (or the molecule, e.g. antibody, comprising said Fc
domain) exhibits less than 50%, preferably less than 20%, more
preferably less than 10% and most preferably less than 5% of the
binding affinity to an Fc receptor, as compared to a native
IgG.sub.1 Fc domain (or a corresponding molecule comprising a
native IgG.sub.1 Fc domain), and/or less than 50%, preferably less
than 20%, more preferably less than 10% and most preferably less
than 5% of the effector function, as compared to a native IgG.sub.1
Fc domain (or a corresponding molecule comprising a native
IgG.sub.1 Fc domain).
[0542] In one embodiment, the Fc domain (or the molecule, e.g.
antibody, comprising said Fc domain) does not substantially bind to
an Fc receptor and/or induce effector function. In a particular
embodiment the Fc receptor is an Fc.gamma. receptor. In one
embodiment the Fc receptor is a human Fc receptor. In one
embodiment the Fc receptor is an activating Fc receptor. In a
specific embodiment the Fc receptor is an activating human
Fc.gamma. receptor, more specifically human Fc.gamma.RIIIa,
Fc.gamma.RI or Fc.gamma.RIIa, most specifically human
Fc.gamma.RIIIa. In one embodiment the effector function is one or
more selected from the group of CDC, ADCC, ADCP, and cytokine
secretion. In a particular embodiment the effector function is
ADCC. In one embodiment the Fc domain exhibits substantially
similar binding affinity to neonatal Fc receptor (FcRn), as
compared to a native IgG.sub.1 Fc domain. Substantially similar
binding to FcRn is achieved when the Fc domain (or the molecule,
e.g. antibody, comprising said Fc domain) exhibits greater than
about 70%, particularly greater than about 80%, more particularly
greater than about 90% of the binding affinity of a native
IgG.sub.1 Fc domain (or the corresponding molecule comprising a
native IgG.sub.1 Fc domain) to FcRn.
[0543] In certain embodiments the Fc domain is engineered to have
reduced binding affinity to an Fc receptor and/or reduced effector
function, as compared to a non-engineered Fc domain. In particular
embodiments, the Fc domain comprises one or more amino acid
mutation that reduces the binding affinity of the Fc domain to an
Fc receptor and/or effector function. Typically, the same one or
more amino acid mutation is present in each of the two subunits of
the Fc domain. In one embodiment the amino acid mutation reduces
the binding affinity of the Fc domain to an Fc receptor. In one
embodiment the amino acid mutation reduces the binding affinity of
the Fc domain to an Fc receptor by at least 2-fold, at least
5-fold, or at least 10-fold. In embodiments where there is more
than one amino acid mutation that reduces the binding affinity of
the Fc domain to the Fc receptor, the combination of these amino
acid mutations may reduce the binding affinity of the Fc domain to
an Fc receptor by at least 10-fold, at least 20-fold, or even at
least 50-fold. In one embodiment the molecule, e.g. antibody,
comprising an engineered Fc domain exhibits less than 20%,
particularly less than 10%, more particularly less than 5% of the
binding affinity to an Fc receptor as compared to a corresponding
molecule comprising a non-engineered Fc domain. In a particular
embodiment the Fc receptor is an Fc.gamma. receptor. In some
embodiments the Fc receptor is a human Fc receptor. In some
embodiments the Fc receptor is an activating Fc receptor. In a
specific embodiment the Fc receptor is an activating human
Fc.gamma. receptor, more specifically human Fc.gamma.RIIIa,
Fc.gamma.RI or Fc.gamma.RIIa, most specifically human
Fc.gamma.RIIIa. Preferably, binding to each of these receptors is
reduced. In some embodiments binding affinity to a complement
component, specifically binding affinity to C1q, is also reduced.
In one embodiment binding affinity to neonatal Fc receptor (FcRn)
is not reduced. Substantially similar binding to FcRn, i.e.
preservation of the binding affinity of the Fc domain to said
receptor, is achieved when the Fc domain (or the molecule, e.g.
antibody, comprising said Fc domain) exhibits greater than about
70% of the binding affinity of a non-engineered form of the Fc
domain (or a corresponding molecule comprising said non-engineered
form of the Fc domain) to FcRn. The Fc domain, or molecule (e.g.
antibody) comprising said Fc domain, may exhibit greater than about
80% and even greater than about 90% of such affinity. In certain
embodiments the Fc domain is engineered to have reduced effector
function, as compared to a non-engineered Fc domain. The reduced
effector function can include, but is not limited to, one or more
of the following: reduced complement dependent cytotoxicity (CDC),
reduced antibody-dependent cell-mediated cytotoxicity (ADCC),
reduced antibody-dependent cellular phagocytosis (ADCP), reduced
cytokine secretion, reduced immune complex-mediated antigen uptake
by antigen-presenting cells, reduced binding to NK cells, reduced
binding to macrophages, reduced binding to monocytes, reduced
binding to polymorphonuclear cells, reduced direct signaling
inducing apoptosis, reduced crosslinking of target-bound
antibodies, reduced dendritic cell maturation, or reduced T cell
priming. In one embodiment the reduced effector function is one or
more selected from the group of reduced CDC, reduced ADCC, reduced
ADCP, and reduced cytokine secretion. In a particular embodiment
the reduced effector function is reduced ADCC. In one embodiment
the reduced ADCC is less than 20% of the ADCC induced by a
non-engineered Fc domain (or a corresponding molecule comprising a
non-engineered Fc domain).
[0544] In one embodiment the amino acid mutation that reduces the
binding affinity of the Fc domain to an Fc receptor and/or effector
function is an amino acid substitution. In one embodiment the Fc
domain comprises an amino acid substitution at a position selected
from the group of E233, L234, L235, N297, P331 and P329 (numberings
according to Kabat EU index). In a more specific embodiment the Fc
domain comprises an amino acid substitution at a position selected
from the group of L234, L235 and P329 (numberings according to
Kabat EU index). In some embodiments the Fc domain comprises the
amino acid substitutions L234A and L235A (numberings according to
Kabat EU index). In one such embodiment, the Fc domain is an
IgG.sub.1 Fc domain, particularly a human IgG.sub.1 Fc domain. In
one embodiment the Fc domain comprises an amino acid substitution
at position P329. In a more specific embodiment the amino acid
substitution is P329A or P329G, particularly P329G (numberings
according to Kabat EU index). In one embodiment the Fc domain
comprises an amino acid substitution at position P329 and a further
amino acid substitution at a position selected from E233, L234,
L235, N297 and P331 (numberings according to Kabat EU index). In a
more specific embodiment the further amino acid substitution is
E233P, L234A, L235A, L235E, N297A, N297D or P331S. In particular
embodiments the Fc domain comprises amino acid substitutions at
positions P329, L234 and L235 (numberings according to Kabat EU
index). In more particular embodiments the Fc domain comprises the
amino acid mutations L234A, L235A and P329G ("P329G LALA"). In one
such embodiment, the Fc domain is an IgG.sub.1 Fc domain,
particularly a human IgG.sub.1 Fc domain. The "P329G LALA"
combination of amino acid substitutions almost completely abolishes
Fc.gamma. receptor (as well as complement) binding of a human
IgG.sub.1 Fc domain, as described in PCT publication no. WO
2012/130831, incorporated herein by reference in its entirety. WO
2012/130831 also describes methods of preparing such mutant Fc
domains and methods for determining its properties such as Fc
receptor binding or effector functions.
[0545] IgG.sub.4 antibodies exhibit reduced binding affinity to Fc
receptors and reduced effector functions as compared to IgG.sub.1
antibodies. Hence, in some embodiments the Fc domain is an
IgG.sub.4 Fc domain, particularly a human IgG.sub.4 Fc domain. In
one embodiment the IgG.sub.4 Fc domain comprises amino acid
substitutions at position S228, specifically the amino acid
substitution S228P (numberings according to Kabat EU index). To
further reduce its binding affinity to an Fc receptor and/or its
effector function, in one embodiment the IgG.sub.4 Fc domain
comprises an amino acid substitution at position L235, specifically
the amino acid substitution L235E (numberings according to Kabat EU
index). In another embodiment, the IgG.sub.4 Fc domain comprises an
amino acid substitution at position P329, specifically the amino
acid substitution P329G (numberings according to Kabat EU index).
In a particular embodiment, the IgG.sub.4 Fc domain comprises amino
acid substitutions at positions S228, L235 and P329, specifically
amino acid substitutions S228P, L235E and P329G (numberings
according to Kabat EU index). Such IgG.sub.4 Fc domain mutants and
their Fc.gamma. receptor binding properties are described in PCT
publication no. WO 2012/130831, incorporated herein by reference in
its entirety.
[0546] In a particular embodiment the Fc domain exhibiting reduced
binding affinity to an Fc receptor and/or reduced effector
function, as compared to a native IgG.sub.1 Fc domain, is a human
IgG.sub.1 Fc domain comprising the amino acid substitutions L234A,
L235A and optionally P329G, or a human IgG.sub.4 Fc domain
comprising the amino acid substitutions S228P, L235E and optionally
P329G (numberings according to Kabat EU index).
[0547] In certain embodiments N-glycosylation of the Fc domain has
been eliminated. In one such embodiment the Fc domain comprises an
amino acid mutation at position N297, particularly an amino acid
substitution replacing asparagine by alanine (N297A) or aspartic
acid (N297D) or glycine (N297G) (numberings according to Kabat EU
index).
[0548] In addition to the Fc domains described hereinabove and in
PCT publication no. WO 2012/130831, Fc domains with reduced Fc
receptor binding and/or effector function also include those with
substitution of one or more of Fc domain residues 238, 265, 269,
270, 297, 327 and 329 (U.S. Pat. No. 6,737,056) (numberings
according to Kabat EU index). Such Fc mutants include Fc mutants
with substitutions at two or more of amino acid positions 265, 269,
270, 297 and 327, including the so-called "DANA" Fc mutant with
substitution of residues 265 and 297 to alanine (U.S. Pat. No.
7,332,581).
[0549] Mutant Fc domains can be prepared by amino acid deletion,
substitution, insertion or modification using genetic or chemical
methods well known in the art. Genetic methods may include
site-specific mutagenesis of the encoding DNA sequence, PCR, gene
synthesis, and the like. The correct nucleotide changes can be
verified for example by sequencing.
[0550] Binding to Fc receptors can be easily determined e.g. by
ELISA, or by Surface Plasmon Resonance (SPR) using standard
instrumentation such as a BIAcore instrument (GE Healthcare), and
Fc receptors such as may be obtained by recombinant expression.
Alternatively, binding affinity of Fc domains or molecules
comprising an Fc domain for Fc receptors may be evaluated using
cell lines known to express particular Fc receptors, such as human
NK cells expressing Fc.gamma.IIIa receptor.
[0551] Effector function of an Fc domain, or a molecule (e.g. an
antibody) comprising an Fc domain, can be measured by methods known
in the art. A suitable assay for measuring ADCC is described
herein. Other examples of in vitro assays to assess ADCC activity
of a molecule of interest are described in U.S. Pat. No. 5,500,362;
Hellstrom et al. Proc Natl Acad Sci USA 83, 7059-7063 (1986) and
Hellstrom et al., Proc Natl Acad Sci USA 82, 1499-1502 (1985); U.S.
Pat. No. 5,821,337; Bruggemann et al., J Exp Med 166, 1351-1361
(1987). Alternatively, non-radioactive assays methods may be
employed (see, for example, ACTI.TM. non-radioactive cytotoxicity
assay for flow cytometry (CellTechnology, Inc. Mountain View,
Calif.); and CytoTox 96.RTM. non-radioactive cytotoxicity assay
(Promega, Madison, Wis.)). Useful effector cells for such assays
include peripheral blood mononuclear cells (PBMC) and Natural
Killer (NK) cells. Alternatively, or additionally, ADCC activity of
the molecule of interest may be assessed in vivo, e.g. in a animal
model such as that disclosed in Clynes et al., Proc Natl Acad Sci
USA 95, 652-656 (1998).
[0552] In some embodiments, binding of the Fc domain to a
complement component, specifically to C1q, is reduced. Accordingly,
in some embodiments wherein the Fc domain is engineered to have
reduced effector function, said reduced effector function includes
reduced CDC. C1q binding assays may be carried out to determine
whether the Fc domain, or molecule (e.g. antibody) comprising the
Fc domain, is able to bind C1q and hence has CDC activity. See
e.g., C1q and C3c binding ELISA in WO 2006/029879 and WO
2005/100402. To assess complement activation, a CDC assay may be
performed (see, for example, Gazzano-Santoro et al., J Immunol
Methods 202, 163 (1996); Cragg et al., Blood 101, 1045-1052 (2003);
and Cragg and Glennie, Blood 103, 2738-2743 (2004)).
[0553] Antigen Binding Moieties
[0554] The antibody comprised in the therapeutic agent may be
bispecific, i.e. it comprises at least two antigen binding moieties
capable of specific binding to two distinct antigenic determinants.
According to particular embodiments, the antigen binding moieties
are Fab molecules (i.e. antigen binding domains composed of a heavy
and a light chain, each comprising a variable and a constant
domain). In one embodiment said Fab molecules are human. In another
embodiment said Fab molecules are humanized. In yet another
embodiment said Fab molecules comprise human heavy and light chain
constant domains.
[0555] In some embodiments, at least one of the antigen binding
moieties is a crossover Fab molecule. Such modification reduces
mispairing of heavy and light chains from different Fab molecules,
thereby improving the yield and purity of the antibody in
recombinant production. In a particular crossover Fab molecule
useful for the antibody, the variable domains of the Fab light
chain and the Fab heavy chain (VL and VH, respectively) are
exchanged. Even with this domain exchange, however, the preparation
of the antibody may comprise certain side products due to a
so-called Bence Jones-type interaction between mispaired heavy and
light chains (see Schaefer et al, PNAS, 108 (2011) 11187-11191). To
further reduce mispairing of heavy and light chains from different
Fab molecules and thus increase the purity and yield of the desired
antibody, charged amino acids with opposite charges may be
introduced at specific amino acid positions in the CH1 and CL
domains of either the Fab molecule(s) specifically binding to a
target cell antigen, or the Fab molecule specifically binding to an
activating T cell antigen. Charge modifications are made either in
the conventional Fab molecule(s) comprised in the antibody (such as
shown e.g. in FIG. 6 A-C, G-J), or in the VH/VL crossover Fab
molecule(s) comprised in the antibody (such as shown e.g. in FIG. 6
D-F, K-N) (but not in both). In particular embodiments, the charge
modifications are made in the conventional Fab molecule(s)
comprised in the antibody (which in particular embodiments
specifically bind(s) to the target cell antigen).
[0556] In a particular embodiment according to the invention, the
antibody is capable of simultaneous binding to a target cell
antigen, particularly a tumor cell antigen, and an activating T
cell antigen, particularly CD3. In one embodiment, the antibody is
capable of crosslinking a T cell and a target cell by simultaneous
binding to a target cell antigen and an activating T cell antigen.
In an even more particular embodiment, such simultaneous binding
results in lysis of the target cell, particularly a tumor cell. In
one embodiment, such simultaneous binding results in activation of
the T cell. In other embodiments, such simultaneous binding results
in a cellular response of a T lymphocyte, particularly a cytotoxic
T lymphocyte, selected from the group of: proliferation,
differentiation, cytokine secretion, cytotoxic effector molecule
release, cytotoxic activity, and expression of activation markers.
In one embodiment, binding of the antibody to the activating T cell
antigen, particularly CD3, without simultaneous binding to the
target cell antigen does not result in T cell activation.
[0557] In one embodiment, the antibody is capable of re-directing
cytotoxic activity of a T cell to a target cell. In a particular
embodiment, said re-direction is independent of MHC-mediated
peptide antigen presentation by the target cell and and/or
specificity of the T cell.
[0558] Particularly, a T cell according to any of the embodiments
of the invention is a cytotoxic T cell. In some embodiments the T
cell is a CD4.sup.+ or a CD8.sup.+ T cell, particularly a CD8.sup.+
T cell.
[0559] (i) Activating T Cell Antigen Binding Moiety
[0560] In some embodiments, an antibody comprised in the
therapeutic agent, particularly a bispecific antibody, comprises at
least one antigen binding moiety, particularly a Fab molecule,
which specifically binds to an activating T cell antigen (also
referred to herein as an "activating T cell antigen binding moiety,
or activating T cell antigen binding Fab molecule"). In a
particular embodiment, the antibody comprises not more than one
antigen binding moiety capable of specific binding to an activating
T cell antigen. In one embodiment, the antibody provides monovalent
binding to the activating T cell antigen.
[0561] In particular embodiments, the antigen binding moiety which
specifically binds an activating T cell antigen is a crossover Fab
molecule as described herein, i.e. a Fab molecule wherein the
variable domains VH and VL or the constant domains CH1 and CL of
the Fab heavy and light chains are exchanged/replaced by each
other. In such embodiments, the antigen binding moiety(ies) which
specifically binds a target cell antigen is preferably a
conventional Fab molecule. In embodiments where there is more than
one antigen binding moiety, particularly Fab molecule, which
specifically binds to a target cell antigen comprised in the
antibody, the antigen binding moiety which specifically binds to an
activating T cell antigen preferably is a crossover Fab molecule
and the antigen binding moieties which specifically bind to a
target cell antigen are conventional Fab molecules.
[0562] In alternative embodiments, the antigen binding moiety which
specifically binds an activating T cell antigen is a conventional
Fab molecule. In such embodiments, the antigen binding moiety(ies)
which specifically binds a target cell antigen is a crossover Fab
molecule as described herein, i.e. a Fab molecule wherein the
variable domains VH and VL or the constant domains CH1 and CL of
the Fab heavy and light chains are exchanged/replaced by each
other.
[0563] In one embodiment, the activating T cell antigen is selected
from the group consisting of CD3, CD28, CD137 (also known as
4-1BB), CD40, CD226, OX40, CD27 HVEM, and CD127.
[0564] In a particular embodiment, the activating T cell antigen is
CD3, particularly human CD3 (SEQ ID NO: 115) or cynomolgus CD3 (SEQ
ID NO: 116), most particularly human CD3. In a particular
embodiment the activating T cell antigen binding moiety is
cross-reactive for (i.e. specifically binds to) human and
cynomolgus CD3. In some embodiments, the activating T cell antigen
is the epsilon subunit of CD3 (CD3 epsilon).
[0565] In some embodiments, the activating T cell antigen binding
moiety specifically binds to CD3, particularly CD3 epsilon, and
comprises at least one heavy chain complementarity determining
region (CDR) selected from the group consisting of SEQ ID NO: 32,
SEQ ID NO: 33 and SEQ ID NO: 34 and at least one light chain CDR
selected from the group of SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO:
37.
[0566] In one embodiment the CD3 binding antigen binding moiety,
particularly Fab molecule, comprises a heavy chain variable region
comprising the heavy chain CDR1 of SEQ ID NO: 32, the heavy chain
CDR2 of SEQ ID NO: 33, the heavy chain CDR3 of SEQ ID NO: 34, and a
light chain variable region comprising the light chain CDR1 of SEQ
ID NO: 35, the light chain CDR2 of SEQ ID NO: 36, and the light
chain CDR3 of SEQ ID NO: 37.
[0567] In one embodiment the CD3 binding antigen binding moiety,
particularly Fab molecule, comprises a heavy chain variable region
sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%
identical to SEQ ID NO: 38 and a light chain variable region
sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%
identical to SEQ ID NO: 39.
[0568] In one embodiment the CD3 binding antigen binding moiety,
particularly Fab molecule, comprises a heavy chain variable region
comprising the amino acid sequence of SEQ ID NO: 38 and a light
chain variable region comprising the amino acid sequence of SEQ ID
NO: 39.
[0569] In one embodiment the CD3 binding antigen binding moiety,
particularly Fab molecule, comprises the heavy chain variable
region sequence of SEQ ID NO: 38 and the light chain variable
region sequence of SEQ ID NO: 39.
[0570] In some embodiments, the activating T cell antigen binding
moiety specifically binds to CD3, particularly CD3 epsilon, and
comprises at least one heavy chain HVR selected from the group
consisting of SEQ ID NO: 120, SEQ ID NO: 121 and SEQ ID NO: 122 and
at least one light chain HVR selected from the group of SEQ ID NO:
123, SEQ ID NO: 124, SEQ ID NO: 125.
[0571] In one embodiment the CD3 binding antigen binding moiety,
particularly Fab molecule, comprises a heavy chain variable region
comprising the heavy chain HVR 1 (H1-HVR) of SEQ ID NO: 120, the
H2-HVR of SEQ ID NO: 121, and the H3-HVR of SEQ ID NO: 122; and a
light chain variable region comprising the light chain HVR 1
(L1-HVR) of SEQ ID NO: 123, the L2-HVR of SEQ ID NO: 124 and the
L3-HVR of SEQ ID NO: 125.
[0572] In one embodiment the CD3 binding antigen binding moiety,
particularly Fab molecule, comprises a heavy chain variable region
sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%
identical to SEQ ID NO: 126 and a light chain variable region
sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%
identical to SEQ ID NO: 127.
[0573] In one embodiment the CD3 binding antigen binding moiety,
particularly Fab molecule, comprises a heavy chain variable region
comprising the amino acid sequence of SEQ ID NO: 126 and a light
chain variable region comprising the amino acid sequence of SEQ ID
NO: 127.
[0574] In one embodiment the CD3 binding antigen binding moiety,
particularly Fab molecule, comprises the heavy chain variable
region sequence of SEQ ID NO: 126 and the light chain variable
region sequence of SEQ ID NO: 127.
[0575] (ii) Target Cell Antigen Binding Moiety
[0576] In some embodiments, an antibody comprised in the
therapeutic agent, particularly a bispecific antibody, comprises at
least one antigen binding moiety, particularly a Fab molecule,
which specifically binds to a target cell antigen. In certain
embodiments, the antibody comprises two antigen binding moieties,
particularly Fab molecules, which specifically bind to a target
cell antigen. In a particular such embodiment, each of these
antigen binding moieties specifically binds to the same antigenic
determinant. In an even more particular embodiment, all of these
antigen binding moieties are identical, i.e. they comprise the same
amino acid sequences including the same amino acid substitutions in
the CH1 and CL domain as described herein (if any). In one
embodiment, the antibody comprises an immunoglobulin molecule which
specifically binds to a target cell antigen. In one embodiment the
antibody comprises not more than two antigen binding moieties,
particularly Fab molecules, which specifically bind to a target
cell antigen.
[0577] In particular embodiments, the antigen binding moiety(ies)
which specifically bind to a target cell antigen is/are a
conventional Fab molecule. In such embodiments, the antigen binding
moiety(ies) which specifically binds an activating T cell antigen
is a crossover Fab molecule as described herein, i.e. a Fab
molecule wherein the variable domains VH and VL or the constant
domains CH1 and CL of the Fab heavy and light chains are
exchanged/replaced by each other.
[0578] In alternative embodiments, the antigen binding moiety(ies)
which specifically bind to a target cell antigen is/are a crossover
Fab molecule as described herein, i.e. a Fab molecule wherein the
variable domains VH and VL or the constant domains CH1 and CL of
the Fab heavy and light chains are exchanged/replaced by each
other. In such embodiments, the antigen binding moiety(ies) which
specifically binds an activating T cell antigen is a conventional
Fab molecule.
[0579] The target cell antigen binding moiety is able to direct the
antibody to a target site, for example to a specific type of tumor
cell that expresses the target cell antigen.
[0580] In one embodiment, the target cell antigen is CEA,
particularly human CEA.
[0581] In one embodiment, the antigen binding moiety, particularly
Fab molecule, which specifically binds to CEA comprises a heavy
chain variable region comprising the heavy chain complementarity
determining region (CDR) 1 of SEQ ID NO: 14, the heavy chain CDR 2
of SEQ ID NO: 15, and the heavy chain CDR 3 of SEQ ID NO: 16, and a
light chain variable region comprising the light chain CDR 1 of SEQ
ID NO: 17, the light chain CDR 2 of SEQ ID NO: 18 and the light
chain CDR 3 of SEQ ID NO: 19. In a further embodiment, the antigen
binding moiety, particularly Fab molecule, which specifically binds
to CEA comprises a heavy chain variable region that is at least
95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO:
20, and a light chain variable region that is at least 95%, 96%,
97%, 98%, or 99% identical to the sequence of SEQ ID NO: 21. In
still a further embodiment, the antigen binding moiety,
particularly Fab molecule, which specifically binds to CEA
comprises the heavy chain variable region sequence of SEQ ID NO:
20, and the light chain variable region sequence of SEQ ID NO:
21.
[0582] In one embodiment, the antigen binding moiety, particularly
Fab molecule, which specifically binds to CEA comprises a heavy
chain variable region comprising the heavy chain complementarity
determining region (CDR) 1 of SEQ ID NO: 136, the heavy chain CDR 2
of SEQ ID NO: 137, and the heavy chain CDR 3 of SEQ ID NO: 138, and
a light chain variable region comprising the light chain CDR 1 of
SEQ ID NO: 139, the light chain CDR 2 of SEQ ID NO: 140 and the
light chain CDR 3 of SEQ ID NO: 141. In a further embodiment, the
antigen binding moiety, particularly Fab molecule, which
specifically binds to CEA comprises a heavy chain variable region
that is at least 95%, 96%, 97%, 98%, or 99% identical to the
sequence of SEQ ID NO: 142, and a light chain variable region that
is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of
SEQ ID NO: 143. In still a further embodiment, the antigen binding
moiety, particularly Fab molecule, which specifically binds to CEA
comprises the heavy chain variable region sequence of SEQ ID NO:
142, and the light chain variable region sequence of SEQ ID NO:
143.
[0583] In one embodiment, the target cell antigen is a B-cell
antigen, particularly a malignant B-cell antigen. In one
embodiment, the target cell antigen is a cell surface antigen. In
one embodiment the target cell antigen is selected from the group
consisting of CD20, CD19, CD22, ROR-1, CD37 and CD5.
[0584] In one embodiment, the target cell antigen is CD20,
particularly human CD20.
[0585] In one embodiment, the antigen binding moiety, particularly
Fab molecule, which specifically binds to CD20 comprises a heavy
chain variable region comprising the heavy chain complementarity
determining region (CDR) 1 of SEQ ID NO: 4, the heavy chain CDR 2
of SEQ ID NO: 5, and the heavy chain CDR 3 of SEQ ID NO: 6, and a
light chain variable region comprising the light chain CDR 1 of SEQ
ID NO: 7, the light chain CDR 2 of SEQ ID NO: 8 and the light chain
CDR 3 of SEQ ID NO: 9. In a further embodiment, the antigen binding
moiety, particularly Fab molecule, which specifically binds to CD20
comprises a heavy chain variable region that is at least 95%, 96%,
97%, 98%, or 99% identical to the sequence of SEQ ID NO: 10, and a
light chain variable region that is at least 95%, 96%, 97%, 98%, or
99% identical to the sequence of SEQ ID NO: 11. In still a further
embodiment, the antigen binding moiety, particularly Fab molecule,
which specifically binds to CD20 comprises the heavy chain variable
region sequence of SEQ ID NO: 10, and the light chain variable
region sequence of SEQ ID NO: 11.
[0586] In one embodiment, the antigen binding moiety, particularly
Fab molecule, which specifically binds to CD20 comprises a heavy
chain variable region comprising the heavy chain HVR 1 (H1-HVR) of
SEQ ID NO: 128, the H2-HVR of SEQ ID NO: 129, and the H3-HVR of SEQ
ID NO: 130; and a light chain variable region comprising the light
chain HVR 1 (L1-HVR) of SEQ ID NO: 131, the L2-HVR of SEQ ID NO:
132 and the L3-HVR of SEQ ID NO: 133. In a further embodiment, the
antigen binding moiety, particularly Fab molecule, which
specifically binds to CD20 comprises a heavy chain variable region
that is at least 95%, 96%, 97%, 98%, or 99% identical to the
sequence of SEQ ID NO: 134, and a light chain variable region that
is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of
SEQ ID NO: 135. In still a further embodiment, the antigen binding
moiety, particularly Fab molecule, which specifically binds to CD20
comprises the heavy chain variable region sequence of SEQ ID NO:
134, and the light chain variable region sequence of SEQ ID NO:
135.
[0587] In one embodiment, the target cell antigen is CD19,
particularly human CD19.
[0588] In one embodiment, the antigen binding moiety, particularly
Fab molecule, which specifically binds to CD19 comprises a heavy
chain variable region comprising the heavy chain complementarity
determining region (CDR) 1 of SEQ ID NO: 48, the heavy chain CDR 2
of SEQ ID NO: 49, and the heavy chain CDR 3 of SEQ ID NO: 50, and a
light chain variable region comprising the light chain CDR 1 of SEQ
ID NO: 51, the light chain CDR 2 of SEQ ID NO: 52 and the light
chain CDR 3 of SEQ ID NO: 53. In a further embodiment, the antigen
binding moiety, particularly Fab molecule, which specifically binds
to CD19 comprises a heavy chain variable region that is at least
95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO:
54, and a light chain variable region that is at least 95%, 96%,
97%, 98%, or 99% identical to the sequence of SEQ ID NO: 55. In
still a further embodiment, the antigen binding moiety,
particularly Fab molecule, which specifically binds to CD19
comprises the heavy chain variable region sequence of SEQ ID NO:
54, and the light chain variable region sequence of SEQ ID NO:
55.
[0589] In another embodiment, the antigen binding moiety,
particularly Fab molecule, which specifically binds to CD19
comprises a heavy chain variable region comprising the heavy chain
complementarity determining region (CDR) 1 of SEQ ID NO: 59, the
heavy chain CDR 2 of SEQ ID NO: 60, and the heavy chain CDR 3 of
SEQ ID NO: 61, and a light chain variable region comprising the
light chain CDR 1 of SEQ ID NO: 62, the light chain CDR 2 of SEQ ID
NO: 63 and the light chain CDR 3 of SEQ ID NO: 64. In a further
embodiment, the antigen binding moiety, particularly Fab molecule,
which specifically binds to CD19 comprises a heavy chain variable
region that is at least 95%, 96%, 97%, 98%, or 99% identical to the
sequence of SEQ ID NO: 65, and a light chain variable region that
is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of
SEQ ID NO: 66. In still a further embodiment, the antigen binding
moiety, particularly Fab molecule, which specifically binds to CD19
comprises the heavy chain variable region sequence of SEQ ID NO:
65, and the light chain variable region sequence of SEQ ID NO:
66.
[0590] In another embodiment, the antigen binding moiety,
particularly Fab molecule, which specifically binds to CD19
comprises [0591] (i) a heavy chain variable region comprising the
heavy chain complementarity determining region (CDR) 1 of SEQ ID
NO: 67, the heavy chain CDR 2 of SEQ ID NO: 68, and the heavy chain
CDR 3 of SEQ ID NO: 69, and a light chain variable region
comprising the light chain CDR 1 of SEQ ID NO: 70, the light chain
CDR 2 of SEQ ID NO: 71 and the light chain CDR 3 of SEQ ID NO: 72;
[0592] (ii) a heavy chain variable region comprising the heavy
chain complementarity determining region (CDR) 1 of SEQ ID NO: 75,
the heavy chain CDR 2 of SEQ ID NO: 76, and the heavy chain CDR 3
of SEQ ID NO: 77, and a light chain variable region comprising the
light chain CDR 1 of SEQ ID NO: 78, the light chain CDR 2 of SEQ ID
NO: 79 and the light chain CDR 3 of SEQ ID NO: 80; [0593] (iii) a
heavy chain variable region comprising the heavy chain
complementarity determining region (CDR) 1 of SEQ ID NO: 83, the
heavy chain CDR 2 of SEQ ID NO: 84, and the heavy chain CDR 3 of
SEQ ID NO: 85, and a light chain variable region comprising the
light chain CDR 1 of SEQ ID NO: 86, the light chain CDR 2 of SEQ ID
NO: 87 and the light chain CDR 3 of SEQ ID NO: 88; [0594] (iv) a
heavy chain variable region comprising the heavy chain
complementarity determining region (CDR) 1 of SEQ ID NO: 91, the
heavy chain CDR 2 of SEQ ID NO: 92, and the heavy chain CDR 3 of
SEQ ID NO: 93, and a light chain variable region comprising the
light chain CDR 1 of SEQ ID NO: 94, the light chain CDR 2 of SEQ ID
NO: 95 and the light chain CDR 3 of SEQ ID NO: 96; [0595] (v) a
heavy chain variable region comprising the heavy chain
complementarity determining region (CDR) 1 of SEQ ID NO: 99, the
heavy chain CDR 2 of SEQ ID NO: 100, and the heavy chain CDR 3 of
SEQ ID NO: 101, and a light chain variable region comprising the
light chain CDR 1 of SEQ ID NO: 102, the light chain CDR 2 of SEQ
ID NO: 103 and the light chain CDR 3 of SEQ ID NO: 104; or [0596]
(vi) a heavy chain variable region comprising the heavy chain
complementarity determining region (CDR) 1 of SEQ ID NO: 107, the
heavy chain CDR 2 of SEQ ID NO: 108, and the heavy chain CDR 3 of
SEQ ID NO: 109, and a light chain variable region comprising the
light chain CDR 1 of SEQ ID NO: 110, the light chain CDR 2 of SEQ
ID NO: 111 and the light chain CDR 3 of SEQ ID NO: 112.
[0597] In a further embodiment, the antigen binding moiety,
particularly Fab molecule, which specifically binds to CD19
comprises [0598] (i) a heavy chain variable region that is at least
95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO:
73, and a light chain variable region that is at least 95%, 96%,
97%, 98%, or 99% identical to the sequence of SEQ ID NO: 74; [0599]
(ii) a heavy chain variable region that is at least 95%, 96%, 97%,
98%, or 99% identical to the sequence of SEQ ID NO: 81, and a light
chain variable region that is at least 95%, 96%, 97%, 98%, or 99%
identical to the sequence of SEQ ID NO: 82; [0600] (iii) a heavy
chain variable region that is at least 95%, 96%, 97%, 98%, or 99%
identical to the sequence of SEQ ID NO: 89, and a light chain
variable region that is at least 95%, 96%, 97%, 98%, or 99%
identical to the sequence of SEQ ID NO: 90; [0601] (iv) a heavy
chain variable region that is at least 95%, 96%, 97%, 98%, or 99%
identical to the sequence of SEQ ID NO: 97, and a light chain
variable region that is at least 95%, 96%, 97%, 98%, or 99%
identical to the sequence of SEQ ID NO: 98; [0602] (v) a heavy
chain variable region that is at least 95%, 96%, 97%, 98%, or 99%
identical to the sequence of SEQ ID NO: 105, and a light chain
variable region that is at least 95%, 96%, 97%, 98%, or 99%
identical to the sequence of SEQ ID NO: 106; or [0603] (vi) a heavy
chain variable region that is at least 95%, 96%, 97%, 98%, or 99%
identical to the sequence of SEQ ID NO: 113, and a light chain
variable region that is at least 95%, 96%, 97%, 98%, or 99%
identical to the sequence of SEQ ID NO: 114.
[0604] In still a further embodiment, the antigen binding moiety,
particularly Fab molecule, which specifically binds to CD19
comprises [0605] (i) the heavy chain variable region sequence of
SEQ ID NO: 73, and the light chain variable region sequence of SEQ
ID NO: 74; [0606] (ii) the heavy chain variable region sequence of
SEQ ID NO: 81, and the light chain variable region sequence of SEQ
ID NO: 82; [0607] (iii) the heavy chain variable region sequence of
SEQ ID NO: 89, and the light chain variable region sequence of SEQ
ID NO: 90; [0608] (iv) the heavy chain variable region sequence of
SEQ ID NO: 97, and the light chain variable region sequence of SEQ
ID NO: 98; [0609] (v) the heavy chain variable region sequence of
SEQ ID NO: 105, and the light chain variable region sequence of SEQ
ID NO: 106; or [0610] (vi) the heavy chain variable region sequence
of SEQ ID NO: 113, and the light chain variable region sequence of
SEQ ID NO: 114.
[0611] Charge Modifications
[0612] An antibody, particularly a multispecific antibody,
comprised in the therapeutic agent may comprise amino acid
substitutions in Fab molecules comprised therein which are
particularly efficient in reducing mispairing of light chains with
non-matching heavy chains (Bence-Jones-type side products), which
can occur in the production of Fab-based bi-/multispecific antigen
binding molecules with a VH/VL exchange in one (or more, in case of
molecules comprising more than two antigen-binding Fab molecules)
of their binding arms (see also PCT publication no. WO 2015/150447,
particularly the examples therein, incorporated herein by reference
in its entirety).
[0613] Accordingly, in particular embodiments, an antibody
comprised in the therapeutic agent comprises
[0614] (a) a first Fab molecule which specifically binds to a first
antigen
[0615] (b) a second Fab molecule which specifically binds to a
second antigen, and wherein the variable domains VL and VH of the
Fab light chain and the Fab heavy chain are replaced by each
other,
[0616] wherein the first antigen is an activating T cell antigen
and the second antigen is a target cell antigen, or the first
antigen is a target cell antigen and the second antigen is an
activating T cell antigen; and
[0617] wherein [0618] i) in the constant domain CL of the first Fab
molecule under a) the amino acid at position 124 is substituted by
a positively charged amino acid (numbering according to Kabat), and
wherein in the constant domain CH1 of the first Fab molecule under
a) the amino acid at position 147 or the amino acid at position 213
is substituted by a negatively charged amino acid (numbering
according to Kabat EU index); or [0619] ii) in the constant domain
CL of the second Fab molecule under b) the amino acid at position
124 is substituted by a positively charged amino acid (numbering
according to Kabat), and wherein in the constant domain CH1 of the
second Fab molecule under b) the amino acid at position 147 or the
amino acid at position 213 is substituted by a negatively charged
amino acid (numbering according to Kabat EU index).
[0620] The antibody does not comprise both modifications mentioned
under i) and ii). The constant domains CL and CH1 of the second Fab
molecule are not replaced by each other (i.e. remain
unexchanged).
[0621] In one embodiment of the antibody, in the constant domain CL
of the first Fab molecule under a) the amino acid at position 124
is substituted independently by lysine (K), arginine (R) or
histidine (H) (numbering according to Kabat) (in one preferred
embodiment independently by lysine (K) or arginine (R)), and in the
constant domain CH1 of the first Fab molecule under a) the amino
acid at position 147 or the amino acid at position 213 is
substituted independently by glutamic acid (E), or aspartic acid
(D) (numbering according to Kabat EU index).
[0622] In a further embodiment, in the constant domain CL of the
first Fab molecule under a) the amino acid at position 124 is
substituted independently by lysine (K), arginine (R) or histidine
(H) (numbering according to Kabat), and in the constant domain CH1
of the first Fab molecule under a) the amino acid at position 147
is substituted independently by glutamic acid (E), or aspartic acid
(D) (numbering according to Kabat EU index).
[0623] In a particular embodiment, in the constant domain CL of the
first Fab molecule under a) the amino acid at position 124 is
substituted independently by lysine (K), arginine (R) or histidine
(H) (numbering according to Kabat) (in one preferred embodiment
independently by lysine (K) or arginine (R)) and the amino acid at
position 123 is substituted independently by lysine (K), arginine
(R) or histidine (H) (numbering according to Kabat) (in one
preferred embodiment independently by lysine (K) or arginine (R)),
and in the constant domain CH1 of the first Fab molecule under a)
the amino acid at position 147 is substituted independently by
glutamic acid (E), or aspartic acid (D) (numbering according to
Kabat EU index) and the amino acid at position 213 is substituted
independently by glutamic acid (E), or aspartic acid (D) (numbering
according to Kabat EU index).
[0624] In a more particular embodiment, in the constant domain CL
of the first Fab molecule under a) the amino acid at position 124
is substituted by lysine (K) (numbering according to Kabat) and the
amino acid at position 123 is substituted by lysine (K) or arginine
(R) (numbering according to Kabat), and in the constant domain CH1
of the first Fab molecule under a) the amino acid at position 147
is substituted by glutamic acid (E) (numbering according to Kabat
EU index) and the amino acid at position 213 is substituted by
glutamic acid (E) (numbering according to Kabat EU index).
[0625] In an even more particular embodiment, in the constant
domain CL of the first Fab molecule under a) the amino acid at
position 124 is substituted by lysine (K) (numbering according to
Kabat) and the amino acid at position 123 is substituted by
arginine (R) (numbering according to Kabat), and in the constant
domain CH1 of the first Fab molecule under a) the amino acid at
position 147 is substituted by glutamic acid (E) (numbering
according to Kabat EU index) and the amino acid at position 213 is
substituted by glutamic acid (E) (numbering according to Kabat EU
index).
[0626] In particular embodiments, the constant domain CL of the
first Fab molecule under a) is of kappa isotype.
[0627] Alternatively, the amino acid substitutions according to the
above embodiments may be made in the constant domain CL and the
constant domain CH1 of the second Fab molecule under b) instead of
in the constant domain CL and the constant domain CH1 of the first
Fab molecule under a). In particular such embodiments, the constant
domain CL of the second Fab molecule under b) is of kappa
isotype.
[0628] The antibody may further comprise a third Fab molecule which
specifically binds to the first antigen. In particular embodiments,
said third Fab molecule is identical to the first Fab molecule
under a). In these embodiments, the amino acid substitutions
according to the above embodiments will be made in the constant
domain CL and the constant domain CH1 of each of the first Fab
molecule and the third Fab molecule. Alternatively, the amino acid
substitutions according to the above embodiments may be made in the
constant domain CL and the constant domain CH1 of the second Fab
molecule under b), but not in the constant domain CL and the
constant domain CH1 of the first Fab molecule and the third Fab
molecule.
[0629] In particular embodiments, the antibody further comprises an
Fc domain composed of a first and a second subunit capable of
stable association.
[0630] Treatment Regimen
[0631] According to the invention, the Type II anti-CD20 antibody
and the therapeutic agent may be administered in various ways (e.g.
with regard to the route of administration, dose and/or timing), as
long as the Type II anti-CD20 antibody is administered prior to the
therapeutic agent and that the administration of the Type II
anti-CD20 antibody has effectively induced a reduction of the
number of B cells in the treated subject by the time the
therapeutic agent is administered.
[0632] Without wishing to be bound by theory, the reduction of the
number of B cells in the subject prior to administration of the
therapeutic agent will reduce or prevent the formation of anti-drug
antibodies (ADAs) to the therapeutic agent and thus reduce or
prevent a loss of efficacy of the therapeutic agent and/or adverse
events in the subject associated with ADAs, and/or will reduce or
prevent cytokine release associated with administration of the
therapeutic agent and thus reduce or prevent adverse events (such
as IRRs) in the subject associated with the administration of the
therapeutic agent.
[0633] In one embodiment, the treatment regimen effectively reduces
the formation of anti-drug antibodies (ADAs) in the subject in
response to the administration of the therapeutic agent as compared
to a corresponding treatment regimen without the administration of
the Type II anti-CD20 antibody. In one embodiment, the formation of
ADAs is reduced at least 2-fold, at least 3-fold, at least 4-fold,
at least 5-fold, at least 10-fold, at least 20-fold, at least
50-fold, or at least 100-fold as compared to a corresponding
treatment regimen without the administration of the Type II
anti-CD20 antibody. In one embodiment, the formation of ADAs is
essentially prevented. In one embodiment, the reduction or
prevention of the formation of ADAs is 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 days, or 1 month,
2 months, 3 months, 4 months, 5 months, 6 months, 12 months, or
more, after administration of the therapeutic agent. In one
embodiment, the reduction or prevention of ADA is about 2 months
after administration of the therapeutic agent.
[0634] In one embodiment, the ADA titer in the subject after
administration of the therapeutic agent does not exceed the ADA
titer in the subject prior to administration of the therapeutic
agent. In one embodiment, the ADA titer in the subject after
administration of the therapeutic agent does not exceed the ADA
titer in the subject prior to administration of the therapeutic
agent by more than 1.1-fold, more than 1.2-fold, more than
1.5-fold, more than 2-fold, more than 3-fold, more than 4-fold,
more than 5-fold, or more than 10-fold. In one embodiment, the ADA
titer in the subject after administration of the therapeutic agent
is increased less than 1.1-fold, less than 1.2-fold, less than
1.5-fold, less than 2-fold, less than 3-fold, less than 4-fold,
less than 5-fold, or less than 10-fold, as compared to the ADA
titer in the subject prior to administration of the therapeutic
agent. In one embodiment, the ADA titer in the subject after
administration of the therapeutic agent is the ADA titer at 1, 2,
3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or
21 days, or 1 month, 2 months, 3 months, 4 months, 5 months, 6
months, 12 months, or more, after administration of the therapeutic
agent. In one embodiment, the ADA titer in the subject after
administration of the therapeutic agent is the ADA titer at about 2
months after administration of the therapeutic agent.
[0635] In one embodiment, essentially no ADAs are detectable in the
subject after administration of the therapeutic agent, particularly
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
20 or 21 days, or 1 month, 2 months, 3 months, 4 months, 5 months,
6 months, 12 months, or more, after administration of the
therapeutic agent.
[0636] ADAs can be detected by methods known in the art (see e.g.
Mire-Sluis et al., J Immunol Methods (2004) 289, 1-16; Nencini et
al., Drug Dev Res (2014), 75 Suppl 1, S4-6; Schouwenburg et al.,
Nat Rev Rheumatol (2015) 9, 164-172). An exemplary method to detect
ADAs is a sandwich ELISA, in which the therapeutic agent (e.g. an
antibody) is coated to the assay plate, is exposed to serum of the
treated subject, and the presence of ADAs is detected by labelled
therapeutic agent. Another exemplary method to detect ADAs is an
antigen binding test wherein immunoglobulins from the treated
subject's serum are aggregated on a protein (e.g. Protein A
Sepharose) and the presence of ADAs is detected by labelled
therapeutic agent.
[0637] ADAs can be detected e.g. in a blood sample taken from the
subject. In one embodiment, the ADA titer in the subject (as
measured e.g. in a blood sample taken from the subject) does not
exceed a titer (i.e. highest possible dilution of the sample giving
an assay signal above the assay cut point) of about 10, of about
20, of about 30, of about 40, of about 50, of about 100, of about
200, or about 500, or of about 1000 after the administration of the
therapeutic agent, particularly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 days, or 1 month, 2
months, 3 months, 4 months, 5 months, 6 months, 12 months, or more,
after administration of the therapeutic agent.
[0638] In some embodiments, the treatment regimen increases
efficacy of the therapeutic agent, as compared to a corresponding
treatment regimen without the administration of the Type II
anti-CD20 antibody. In some embodiments, the treatment regimen
increases overall survival of the subject, as compared to a
corresponding treatment regimen without the administration of the
Type II anti-CD20 antibody. In some embodiments, the treatment
regimen increases progression-free survival of the subject, as
compared to a corresponding treatment regimen without the
administration of the Type II anti-CD20 antibody.
[0639] In one embodiment, the treatment regimen effectively reduces
cytokine release in the subject associated with the administration
of the therapeutic agent as compared to a corresponding treatment
regimen without the administration of the Type II anti-CD20
antibody. In one embodiment, cytokine release is reduced at least
2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least
10-fold, at least 20-fold, at least 50-fold, or at least 100-fold
as compared to a corresponding treatment regimen without the
administration of the Type II anti-CD20 antibody. In one
embodiment, cytokine release is essentially prevented. In one
embodiment, the reduction or prevention of cytokine release is 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
21, 22, 23 or 24 hours after administration of the therapeutic
agent. In one embodiment, the reduction or prevention of cytokine
release is within the first 24 hours after administration of the
therapeutic agent.
[0640] In one embodiment, the cytokine concentration in the subject
(as measured e.g. in a blood sample taken from the subject) after
administration of the therapeutic agent does not exceed the
cytokine concentration in the subject prior to administration of
the therapeutic agent. In one embodiment, the cytokine
concentration in the subject after administration of the
therapeutic agent does not exceed the cytokine concentration in the
subject prior to administration of the therapeutic agent by more
than 1.1-fold, more than 1.2-fold, more than 1.5-fold, more than
2-fold, more than 3-fold, more than 4-fold, more than 5-fold, more
than 10-fold, more than 20-fold, more than 50-fold or more than
100-fold. In one embodiment, the cytokine concentration in the
subject after administration of the therapeutic agent is increased
less than 1.1-fold, less than 1.2-fold, less than 1.5-fold, less
than 2-fold, less than 3-fold, less than 4-fold, less than 5-fold,
less than 10-fold, less than 20-fold, less than 50-fold or less
than 100-fold, as compared to the cytokine concentration in the
subject prior to administration of the therapeutic agent. In one
embodiment, the cytokine concentration in the subject after
administration of the therapeutic agent is the cytokine
concentration at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, 20, 21, 22, 23 or 24 hours after administration of
the therapeutic agent. In one embodiment, the cytokine
concentration in the subject after administration of the
therapeutic agent is the cytokine concentration within the first 24
hours after administration of the therapeutic agent.
[0641] In one embodiment, essentially no increase in the
concentration of cytokines is detectable in the subject after
administration of the therapeutic agent, particularly 1, 2, 3, 4,
5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,
23 or 24 hours after administration of the therapeutic agent.
[0642] Cytokines can be detected by methods known in the art, such
as e.g. ELISA, FACS or Luminex.RTM. assay.
[0643] Cytokines can be detected e.g. in a blood sample taken from
the subject. In one embodiment, the cytokine concentration is the
blood of the subject.
[0644] In some embodiments, the cytokine is one or more cytokine(s)
selected from the group consisting of tumor necrosis factor alpha
(TNF-.alpha.), interferon gamma (IFN-.gamma.), interleukin-6
(IL-6), interleukin-10 (IL-10), interleukin-2 (IL-2) and
interleukin-8 (IL-8), particularly the group consisting of
TNF-.alpha., IFN-.gamma. and IL-6. In some embodiments, the
cytokine is TNF-.alpha.. In some embodiments, the cytokine is
IFN-.gamma.. In some embodiments, the cytokine is IL-6. In some
embodiments, the cytokine is IL-10. In some embodiments, the
cytokine is IL-2. In some embodiments, the cytokine is IL-8.
[0645] In some embodiments, the treatment regimen increases the
safety of the therapeutic agent, as compared to a corresponding
treatment regimen without the administration of the Type II
anti-CD20 antibody. In some embodiments, the treatment regimen
reduces adverse events in the subject, as compared to a
corresponding treatment regimen without the administration of the
Type II anti-CD20 antibody. In some embodiments, the treatment
regimen increases the serum half-life of the therapeutic agent, as
compared to a corresponding treatment regimen without the
administration of the Type II anti-CD20 antibody. In some
embodiments, the treatment regimen reduces toxicity of the
therapeutic agent, as compared to a corresponding treatment regimen
without the administration of the Type II anti-CD20 antibody.
[0646] According to the invention, the period of time between the
administration of the Type II anti-CD20 antibody and the
administration of the therapeutic agent is sufficient for reduction
of the number of B-cells in the subject in response to the
administration of the Type II anti-CD20 antibody.
[0647] In one embodiment, the period of time is 3 days to 21 days,
5 days to 20 days, 7 days to 21 days, 7 days to 14 days, 5 days to
15 days, 7 days to 15 days, 8 days to 15 days, 10 days to 20 days,
10 days to 15 days, 11 days to 14 days, or 12 days to 13 days. In
one embodiment, the period of time is 7 days to 14 days. In a
particular embodiment, the period of time is 10 days to 15 days. In
one embodiment, the period of time is 8 days to 15 days. In one
embodiment, the period of time is 5 days to 10 days. In a
particular embodiment, the period of time is 7 days.
[0648] In one embodiment, the period of time is about 3 days, about
4 days, about 5 days, about 6 days, about 7 days, about 8 days,
about 9 days, about 10 days, about 11 days, about 12 days, about 13
days, about 14 days, about 15 days, about 16 days, about 17 days,
about 18 days, about 19 days, about 20 days, about 21 days, about
22 days, about 23 days, about 24 days, about 25 days, about 26
days, about 27 days, about 28 days, about 29 days, or about 30
days.
[0649] In one embodiment, the period of time is at least 3 days, at
least 4 days, at least 5 days, at least 6 days, at least 7 days, at
least 8 days, at least 9 days, at least 10 days, at least 11 days,
at least 12 days, at least 13 days, at least 14 days, or at least
15 days. In a particular embodiment, the period of time is at least
5 days. In a further particular embodiment, the period of time is
at least 7 days.
[0650] In one embodiment, the period of time is between the last
administration of the Type II anti-CD20 antibody and the (first, if
several) administration of the therapeutic agent. In one
embodiment, no administration of the therapeutic agent is made
during the period of time.
[0651] In a particular embodiment, the reduction of the number of B
cells is in the blood of the subject. In one embodiment, the B
cells are peripheral blood B cells. In one embodiment, the B cells
are normal B cells. In one embodiment, the B cells are malignant
and normal B cells. In one embodiment, the B cells are malignant B
cells.
[0652] In some embodiments, the reduction of B cells is in a tissue
of the subject. In one embodiment, the tissue is a tumor. In one
embodiment, the tissue is a lymph node. In one embodiment, the
tissue is spleen. In one embodiment, the tissue is the marginal
zone of spleen. In one embodiment, the B cells are lymph node B
cells. In one embodiment, the B cells are splenic B cells. In one
embodiment, the B cells are splenic marginal zone B cells. In one
embodiment, the B cells are CD20-positive B cells, i.e. B cells
expressing CD20 on their surface.
[0653] In one embodiment, the reduction of the number of B cells is
a reduction of at least about 10%, at least about 15%, at least
about 20%, at least about 25%, at least about 30%, at least about
35%, at least about 40%, at least about 45%, at least about 50%, at
least about 55%, at least about 60%, at least about 65%, at least
about 70%, at least about 75%, at least about 80%, at least about
85%, at least about 90%, or at least about 95%. In one embodiment,
the reduction of the number of B cells is a complete elimination of
B cells. In a particular embodiment, the reduction of the number of
B cells is a reduction of at least 90%, particularly at least 95%,
of the number of B cells in the (peripheral) blood of the subject.
In one embodiment, the reduction of the number of B cells is a
reduction as compared to the number of B cells in the subject prior
to the (first, if several) administration of the Type II anti-CD20
antibody to the subject.
[0654] The number of B cells in the subject may be determined by
any method known in the art suitable for quantifying B cells in
patient blood or tissue, such as flow cytometric,
immunohistochemical or immunofluorescent methods, using antibodies
against B cell markers such as CD20, CD19, and/or PAX5.
[0655] The number of B cells may also be determined indirectly, by
quantification of protein or mRNA levels of B-cell markers in
patient blood or tissues. Suitable methods known in the art for the
determination of specific protein levels include immunoassay
methods such as enzyme-linked immunosorbent assay (ELISA), or
Western Blot, methods for determination of mRNA levels include for
example quantitative RT-PCR or microarray technologies.
[0656] All the above mentioned methods and technologies are well
known in the art and can be deduced from standard textbooks such as
Lottspeich (Bioanalytik, Spektrum Akademisher Verlag, 1998) or
Sambrook and Russell (Molecular Cloning: A Laboratory Manual, CSH
Press, Cold Spring Harbor, N.Y., U.S.A., 2001).
[0657] In certain embodiments, the reduction of the number of B
cells is determined by quantification of B cells in the blood of
the subject (e.g. in a blood sample taken from the subject). In one
such embodiment, B cells are quantified by flow cytometric
analysis. Flow cytometric methods (FACS) are well known in the art
for the quantification of cells in blood or tissue samples. In
particular, they allow determining the number of cells expressing a
specific antigen (e.g. CD20 and/or CD19) among a defined total
number of cells in a blood or tissue sample (e.g. a blood sample,
or (part of) a tissue biopsy). In one embodiment, B cells are
quantified by flow cytometric analysis using an anti-CD19 antibody
and/or an anti-CD20 antibody.
[0658] In other embodiments, the reduction of the number of B cells
is determined by quantification of B cells in a tissue, e.g. a
tumor, of said individual (e.g. in a tissue biopsy taken from the
subject). In one such embodiment, B cells are quantified by
immunohistochemical or immunofluorescent analysis. In one
embodiment, B cells are quantified by immunohistochemical analysis
using an anti-CD19 antibody, an anti-CD20 antibody and/or an
anti-PAX5 antibody.
[0659] Methods of the present invention can be applied in the
treatment of a variety of diseases, depending on the therapeutic
agent(s) used.
[0660] In certain embodiments the disease to be treated is a
proliferative disorder, particularly cancer. Non-limiting examples
of cancers include bladder cancer, brain cancer, head and neck
cancer, pancreatic cancer, lung cancer, breast cancer, ovarian
cancer, uterine cancer, cervical cancer, endometrial cancer,
esophageal cancer, colon cancer, colorectal cancer, rectal cancer,
gastric cancer, prostate cancer, blood cancer, skin cancer,
squamous cell carcinoma, bone cancer, and kidney cancer. Other cell
proliferation disorders that can be treated using a method of the
present invention include, but are not limited to neoplasms located
in the: abdomen, bone, breast, digestive system, liver, pancreas,
peritoneum, endocrine glands (adrenal, parathyroid, pituitary,
testicles, ovary, thymus, thyroid), eye, head and neck, nervous
system (central and peripheral), lymphatic system, pelvic, skin,
soft tissue, spleen, thoracic region, and urogenital system. Also
included are pre-cancerous conditions or lesions and cancer
metastases. In certain embodiments the cancer is chosen from the
group consisting of renal cell cancer, skin cancer, lung cancer,
colorectal cancer, breast cancer, brain cancer, head and neck
cancer. In some embodiments, the cancer is a solid tumor. In some
embodiments, the cancer expresses the target of the therapeutic
agent, e.g. an antibody. In one embodiment, the cancer expresses
CEA. In another embodiment, the cancer expresses FAP.
[0661] In some embodiments, the disease to be treated is an
inflammatory disorder. In some embodiments, the disease to be
treated is an autoimmune disease (i.e. a non-malignant disease or
disorder arising from and directed against an individual's own
tissues). Examples of autoimmune diseases or disorders include, but
are not limited to, inflammatory responses such as inflammatory
skin diseases including psoriasis and dermatitis (e.g. atopic
dermatitis); responses associated with inflammatory bowel disease
(such as Crohn's disease and ulcerative colitis); dermatitis;
allergic conditions such as eczema and asthma; rheumatoid
arthritis; systemic lupus erythematosus (SLE) (including but not
limited to lupus nephritis, cutaneous lupus); diabetes mellitus
(e.g. type 1 diabetes mellitus or insulin dependent diabetes
mellitus); multiple sclerosis and juvenile onset diabetes. In one
embodiment, the disease is transplant rejection or
graft-versus-host disease.
[0662] In some embodiments, the disease to be treated is an
infectious disease, such as viral infection or a bacterial
infection. In other embodiments, the disease to be treated is
neurological disorder. In still further embodiments, the disease to
be treated is a metabolic disorder.
[0663] In relation aspects of the invention concerned with the
reduction of cytokine release associated with the administration of
a therapeutic agent in a subject, the methods are particularly
useful, in the treatment of B-cell proliferative disorders,
particularly CD20-positive B-cell disorders, where (CD20-positive)
B-cells are present in large quantities (i.e. an increased number
of B-cells is present in the subject suffering from the disorder,
as compared to a healthy subject).
[0664] Thus, in one embodiment, the disease is a B cell
proliferative disorder, particularly a CD20-positive B-cell
disorder.
[0665] In one embodiment, the disease is selected from the group
consisting of Non-Hodgkin lymphoma (NHL), acute lymphocytic
leukemia (ALL), chronic lymphocytic leukemia (CLL), diffuse large
B-cell lymphoma (DLBCL), follicular lymphoma (FL), mantle-cell
lymphoma (MCL), marginal zone lymphoma (MZL), Multiple myeloma (MM)
or Hodgkin lymphoma (HL). In one embodiment, the disease is
selected from the group consisting of Non-Hodgkin lymphoma (NHL),
acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia
(CLL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma
(FL), mantle-cell lymphoma (MCL) and marginal zone lymphoma
(MZL).
[0666] In a particular embodiment, the disease is NHL, particularly
relapsed/refractory (r/r) NHL. In one embodiment, the disease is
DLBCL. In one embodiment, the disease is FL. In one embodiment, the
disease is MCL. In one embodiment, the disease is MZL.
[0667] A skilled artisan readily recognizes that in many cases the
therapeutic agent may not provide a cure but may only provide
partial benefit. In some embodiments, a physiological change having
some benefit is also considered therapeutically beneficial. Thus,
in some embodiments, an amount of therapeutic agent that provides a
physiological change is considered an "effective amount" or a
"therapeutically effective amount".
[0668] The subject, patient, or individual in need of treatment is
typically a mammal, more specifically a human. In certain
embodiments, the subject is a human.
[0669] In some embodiments, in particular in relation aspects of
the invention concerned with the reduction of the formation of
anti-drug antibodies (ADAs) against a therapeutic agent in a
subject, the subject suffers from a locally advanced and/or
metastatic solid tumor and has progressed on or is intolerant to
the standard of care therapy. In one embodiment, the tumor is a
CEA-expressing tumor. In another embodiment, the tumor is a
FAP-expressing tumor. Expression of CEA and/or FAP in a tumor may
be determined for example by immunohistochemical analysis of a
tumor biopsy taken from the subject.
[0670] In other embodiments, in particular in relation aspects of
the invention concerned with the reduction of cytokine release
associated with the administration of a therapeutic agent in a
subject, the subject suffers from a B-cell proliferative disorder,
particularly from Non-Hodgkin lymphoma (NHL), acute lymphocytic
leukemia (ALL), chronic lymphocytic leukemia (CLL), diffuse large
B-cell lymphoma (DLBCL), follicular lymphoma (FL), mantle-cell
lymphoma (MCL), marginal zone lymphoma (MZL), Multiple myeloma (MM)
or Hodgkin lymphoma (HL). In one embodiment, the subject suffers
from relapsed/refractory (r/r) NHL.
[0671] Administration of the Type II Anti-CD20 Antibody
[0672] According to the invention, the period of time between the
administration of the Type II anti-CD20 antibody and the
administration of the therapeutic agent and the dose of the Type II
anti-CD20 antibody are chosen such as to effectively reduce the
number of B cells in the subject prior to administration of the
therapeutic agent.
[0673] The Type II anti-CD20 antibody can be administered by any
suitable means, including parenteral, intrapulmonary, and
intranasal, and, if desired for local treatment, intralesional
administration. Parenteral infusions include intramuscular,
intravenous, intraarterial, intraperitoneal, or subcutaneous
administration. Dosing can be by any suitable route, e.g. by
injections, such as intravenous or subcutaneous injections,
depending in part on whether the administration is brief or
chronic. Various dosing schedules including but not limited to
single or multiple administrations over various time-points, bolus
administration, and pulse infusion are contemplated herein. In one
embodiment, the Type II anti-CD20 antibody is administered
parenterally, particularly intravenously, e.g. by intravenous
infusion.
[0674] The Type II anti-CD20 antibody would be formulated, dosed,
and administered in a fashion consistent with good medical
practice. Factors for consideration in this context include the
particular disorder being treated, the particular mammal being
treated, the clinical condition of the individual patient, the
cause of the disorder, the site of delivery of the agent, the
method of administration, the scheduling of administration, and
other factors known to medical practitioners.
[0675] In one embodiment, the administration of the Type II
anti-CD20 antibody is a single administration. In another
embodiment, the administration of the Type II anti-CD20 antibody is
two or more separate administrations. In one embodiment, the two or
more separate administrations are on two or more consecutive days.
In one embodiment, no further administration of the Type II
anti-CD20 antibody is made to the subject before or after the
administration of the therapeutic agent. In one embodiment, the
administration of the Type II anti-CD20 antibody is a single
administration, or two administrations on two consecutive days, and
no further administration of the Type II anti-CD20 antibody is
made. In one embodiment, the period of time is between the last
administration of the Type II anti-CD20 antibody and the (first, if
several) administration of the therapeutic agent.
[0676] In one embodiment, the administration of the Type II
anti-CD20 antibody is a dose of Type II anti-CD20 antibody
effective for the reduction of B cells in the subject. In one
embodiment, the dose of Type II anti-CD20 antibody is effective in
reducing the number of B cells in the subject within the period of
time between the administration of the Type II anti-CD20 antibody
and the administration of the therapeutic agent. In one embodiment,
the period of time between the administration of the Type II
anti-CD20 antibody and the administration of the therapeutic agent
and the administered dose of Type II anti-CD20 antibody is
sufficient for reduction of the number of B-cells in the subject in
response to the administration of the Type II anti-CD20
antibody.
[0677] In one embodiment, the administration of the Type II
anti-CD20 antibody is a dose of about 2 g Type II anti-CD20
antibody. The dose of about 2 g Type II anti-CD20 antibody may be
administered to the subject as a single administration of about 2
g, or as several administrations, e.g. two administrations of about
1 g each or three administrations of e. g. 100 mg, 900 mg and 1000
mg. In one embodiment, one administration of about 2 g Type II
anti-CD20 antibody is made to the subject. In another embodiment,
two administrations of about 1 g Type II anti-CD20 antibody each
are made to the subject on two consecutive days. In still another
embodiment, three administrations ((i) to (iii)) of (i) about 100
mg Type II anti-CD20 antibody, (ii) about 900 mg Type II anti-CD20
antibody, and (iii) about 1000 mg Type II anti-CD20 antibody are
made to the subject on three consecutive days. In one embodiment,
two administration of about 1 g Type II anti-CD20 antibody are made
to the subject on two consecutive days, 10 days to 15 days before
the administration of the therapeutic agent. In one embodiment, one
administration of about 2 g Type II anti-CD20 antibody is made to
the subject 10 days to 15 days before the administration of the
therapeutic agent. In one embodiment, no further administration of
the Type II anti-CD20 antibody is made to the subject. In one
embodiment, no administration of the therapeutic agent is made to
the subject prior to the administration of the Type II anti-CD20
antibody (at least not within the same course of treatment).
[0678] In one embodiment, the administration of the Type II
anti-CD20 antibody is a dose of about 1000 mg Type II anti-CD20
antibody. The dose of about 1000 mg Type II anti-CD20 antibody may
be administered to the subject as a single administration of about
1000 mg, or as several administrations, e.g. two administrations of
about 500 mg each. In a particular embodiment, one administration
of about 1000 mg Type II anti-CD20 antibody is made to the subject.
In another embodiment, two administrations of about 500 mg Type II
anti-CD20 antibody each are made to the subject on two consecutive
days. In one embodiment, one administration of about 1000 mg Type
II anti-CD20 antibody is made to the subject, 7 days before the
administration of the therapeutic agent. In one embodiment, no
further administration of the Type II anti-CD20 antibody is made to
the subject. In one embodiment, no administration of the
therapeutic agent is made to the subject prior to the
administration of the Type II anti-CD20 antibody (at least not
within the same course of treatment).
[0679] In one embodiment, the treatment regimen further comprises
administration of premedication prior to the administration of the
Type II anti-CD20 antibody. In embodiment the premedication
comprises a corticosteroid (such as e.g. prednisolone,
dexamethasone, or methylprednisolone), paracetamol/acetaminophen,
and/or an anti-histamine (such as e.g. diphenhydramine). In one
embodiment, the premedication is administered at least 60 minutes
prior to the administration of the Type II anti-CD20 antibody.
[0680] In one embodiment, the treatment regimen does not comprise
administration of an immunosuppressive agent other than the Type II
anti-CD20 antibody (and optionally the above-described
premedication) prior to the administration of the therapeutic
agent. In one embodiment, the treatment regimen does not comprise
administration of an agent selected from the group of methotrexate,
azathioprine, 6-mercaptopurine, leflunomide, cyclosporine,
tacrolimus/FK506, mycophenolate mofetil and mycophenolate sodium
prior to the administration of the therapeutic agent. In one
embodiment, the treatment regimen does not comprise administration
of a further antibody in addition to the Type II anti-CD20 antibody
prior to the administration of the therapeutic agent.
[0681] Administration of the Therapeutic Agent
[0682] The therapeutic agent can be administered by any suitable
means, including parenteral, intrapulmonary, and intranasal, and,
if desired for local treatment, intralesional administration. The
methods of the present invention are particularly useful, however,
in relation to therapeutic agents administered by parenteral,
particularly intravenous, infusion. Parenteral infusions include
intramuscular, intravenous, intraarterial, intraperitoneal, or
subcutaneous administration. Dosing can be by any suitable route,
e.g. by injections, such as intravenous or subcutaneous injections,
depending in part on whether the administration is brief or
chronic. Various dosing schedules including but not limited to
single or multiple administrations over various time-points, bolus
administration, and pulse infusion are contemplated herein. In one
embodiment, the therapeutic agent is administered parenterally,
particularly intravenously. In a particular embodiment, the
therapeutic agent is administered by intravenous infusion.
[0683] The therapeutic agent would be formulated, dosed, and
administered in a fashion consistent with good medical practice.
Factors for consideration in this context include the particular
disorder being treated, the particular mammal being treated, the
clinical condition of the individual patient, the cause of the
disorder, the site of delivery of the agent, the method of
administration, the scheduling of administration, and other factors
known to medical practitioners. The therapeutic agent need not be,
but is optionally formulated with one or more agents currently used
to prevent or treat the disorder in question. The effective amount
of such other agents depends on the amount of therapeutic agent
present in the formulation, the type of disorder or treatment, and
other factors discussed above. These are generally used in the same
dosages and with administration routes as described herein, or
about from 1 to 99% of the dosages described herein, or in any
dosage and by any route that is empirically/clinically determined
to be appropriate.
[0684] For the prevention or treatment of disease, the appropriate
dosage of the therapeutic agent (when used alone or in combination
with one or more other additional therapeutic agents) will depend
on the type of disease to be treated, the type of therapeutic
agent, the severity and course of the disease, whether the
therapeutic agent is administered for preventive or therapeutic
purposes, previous therapy, the patient's clinical history and
response to the therapeutic agent, and the discretion of the
attending physician. The therapeutic agent is suitably administered
to the patient at one time or over a series of treatments.
Depending on the type and severity of the disease, about 1 .mu.g/kg
to 15 mg/kg (e.g. 0.1 mg/kg-10 mg/kg) of therapeutic agent can be
an initial candidate dosage for administration to the subject,
whether, for example, by one or more separate administrations, or
by continuous infusion. One typical daily dosage might range from
about 1 .mu.g/kg to 100 mg/kg or more, depending on the factors
mentioned above. For repeated administrations over several days or
longer, depending on the condition, the treatment would generally
be sustained until a desired suppression of disease symptoms
occurs. One exemplary dosage of the therapeutic agent would be in
the range from about 0.05 mg/kg to about 10 mg/kg. Thus, one or
more doses of about 0.5 mg/kg, 2.0 mg/kg, 4.0 mg/kg or 10 mg/kg (or
any combination thereof) may be administered to the subject. Such
doses may be administered intermittently, e.g. every week, every
two weeks, or every three weeks (e.g. such that the subject
receives from about two to about twenty, or e.g. about six doses of
the therapeutic agent). An initial higher loading dose, followed by
one or more lower doses, or an initial lower dose, followed by one
or more higher doses may be administered. An exemplary dosing
regimen comprises administering an initial dose of about 10 mg,
followed by a bi-weekly dose of about 20 mg of the therapeutic
agent. However, other dosage regimens may be useful. The progress
of this therapy is easily monitored by conventional techniques and
assays.
[0685] In one embodiment, the administration of the therapeutic
agent is a single administration. In certain embodiments, the
administration of the therapeutic agent is two or more
administrations. In one such embodiment, the therapeutic agent is
administered every week, every two weeks, or every three weeks,
particularly every two weeks. In one embodiment, the therapeutic
agent is administered in a therapeutically effective amount. In one
embodiment the therapeutic agent is administered at a dose of 10
mg-20 mg. In one embodiment the administration of the therapeutic
agent comprises an initial administration of a dose of about 10 mg
therapeutic agent, and one or more subsequent administrations of a
dose of about 20 mg therapeutic agent. In one embodiment the
therapeutic agent is administered at a dose of about 50 .mu.g/kg,
about 100 .mu.g/kg, about 200 .mu.g/kg, about 300 .mu.g/kg, about
400 .mu.g/kg, about 500 .mu.g/kg, about 600 .mu.g/kg, about 700
.mu.g/kg, about 800 .mu.g/kg, about 900 .mu.g/kg or about 1000
.mu.g/kg. In one embodiment, the therapeutic agent is administered
at a dose which is higher than the dose of the therapeutic agent in
a corresponding treatment regimen without the administration of the
Type II anti-CD20 antibody. In one embodiment the administration of
the therapeutic agent comprises an initial administration of a
first dose of the therapeutic agent, and one or more subsequent
administrations of a second dose the therapeutic agent, wherein the
second dose is higher than the first dose. In one embodiment, the
administration of the therapeutic agent comprises an initial
administration of a first dose of the therapeutic agent, and one or
more subsequent administrations of a second dose the therapeutic
agent, wherein the first dose is not lower than the second
dose.
[0686] In one embodiment, the administration of the therapeutic
agent in the treatment regimen according to the invention is the
first administration of that therapeutic agent to the subject (at
least within the same course of treatment). In one embodiment, no
administration of the therapeutic agent is made to the subject
prior to the administration of the Type II anti-CD20 antibody.
[0687] In the present invention, the therapeutic agent can be used
either alone or in combination with other agents in a therapy. For
instance, the therapeutic agent may be co-administered with at
least one additional therapeutic agent. In certain embodiments, an
additional therapeutic agent is an immunotherapeutic agent. In some
embodiment, the additional therapeutic agent comprises an agent as
described herein in relation to the therapeutic agent. The
invention is particularly useful in relation to combinations of
several therapeutic agents which may induce the formation of ADAs
or cytokine release in a subject when used in a treatment regimen
without the administration of the Type II anti-CD20 antibody. Such
combinations may include various therapeutic agents as described
herein, and may particularly include one or more immunotherapeutic
agents, e.g. for the treatment of cancer (cancer immunotherapy).
Such combination therapies noted above encompass combined
administration (where two or more therapeutic agents are included
in the same or separate formulations), and separate administration,
in which case, administration of the therapeutic agent can occur
prior to, simultaneously, and/or following, administration of an
additional therapeutic agent or agents. In one embodiment,
administration of the therapeutic agent and administration of an
additional therapeutic agent occur within about one month, or
within about one, two or three weeks, or within about one, two,
three, four, five, or six days, of each other.
[0688] Articles of Manufacture
[0689] In another aspect of the invention, an article of
manufacture, e.g. a kit, is provided, containing materials useful
for the treatment, prevention and/or diagnosis of a disease, or for
the reduction of the formation of anti-drug antibodies (ADAs)
and/or the reduction of cytokine release as described herein. The
article of manufacture comprises a container and a label or package
insert on or associated with the container. Suitable containers
include, for example, bottles, vials, syringes, IV solution bags,
etc. The containers may be formed from a variety of materials such
as glass or plastic. The container holds a composition which is by
itself or combined with another composition effective for treating,
preventing and/or diagnosing the condition, or holds a composition
which is effective for reducing the formation of ADAs and/or
cytokine release, and may have a sterile access port (for example
the container may be an intravenous solution bag or a vial having a
stopper pierceable by a hypodermic injection needle). At least one
active agent in the composition is a Type II anti-CD20 antibody or
a therapeutic agent as described herein. The label or package
insert indicates that the composition is used for treating the
condition of choice and/or to reduce the formation of ADAs and/or
cytokine release. Moreover, the article of manufacture may comprise
(a) a first container with a composition contained therein, wherein
the composition comprises a Type II anti-CD20 antibody as described
herein; and (b) a second container with a composition contained
therein, wherein the composition comprises a therapeutic agent as
described herein. The article of manufacture in this embodiment of
the invention may further comprise a package insert indicating that
the compositions can be used to treat a particular condition and/or
to reduce the formation of ADAs and/or cytokine release.
Alternatively, or additionally, the article of manufacture may
further comprise a second (or third) container comprising a
pharmaceutically-acceptable buffer, such as bacteriostatic water
for injection (BWFI), phosphate-buffered saline, Ringer's solution
and dextrose solution. It may further include other materials
desirable from a commercial and user standpoint, including other
buffers, diluents, filters, needles, and syringes.
EMBODIMENTS
[0690] In the following, some of the embodiments of the invention
are listed.
[0691] 1. A method of treating a disease in a subject, the method
comprising a treatment regimen comprising
[0692] (i) administration to the subject of a Type II anti-CD20
antibody,
[0693] and consecutively after a period of time
[0694] (ii) administration to the subject of a therapeutic
agent,
[0695] wherein the period of time between the administration of the
Type II anti-CD20 antibody and the administration of the
therapeutic agent is sufficient for reduction of the number of
B-cells in the subject in response to the administration of the
Type II anti-CD20 antibody.
[0696] 2. The method of embodiment 1, wherein the treatment regimen
effectively reduces the formation of anti-drug antibodies (ADAs) in
the subject in response to the administration of the therapeutic
agent as compared to a corresponding treatment regimen without the
administration of the Type II anti-CD20 antibody.
[0697] 3. A method for reducing the formation of anti-drug
antibodies (ADAs) against a therapeutic agent in a subject,
comprising administration of a Type II anti-CD20 antibody to the
subject prior to administration of the therapeutic agent.
[0698] 4. The method of embodiment 3, wherein the period of time
between the administration of the Type II anti-CD20 antibody and
administration of the therapeutic agent is sufficient for reduction
of the number of B-cells in the subject in response to the
administration of the Type II anti-CD20 antibody.
[0699] 5. The method of any one of the preceding embodiments,
wherein the Type II anti-CD20 antibody comprises a heavy chain
variable region comprising the heavy chain CDR (HCDR) 1 of SEQ ID
NO: 4, the HCDR2 of SEQ ID NO: 5, and the HCDR3 of SEQ ID NO: 6;
and a light chain variable region comprising the light chain CDR
(LCDR) 1 of SEQ ID NO: 7, the LCDR2 of SEQ ID NO: 8 and the LCDR3
of SEQ ID NO: 9.
[0700] 6. The method of any one of the preceding embodiments,
wherein the Type II anti-CD20 antibody comprises the heavy chain
variable region sequence of SEQ ID NO: 10 and the light chain
variable region sequence of SEQ ID NO: 11.
[0701] 7. The method of any one of the preceding embodiments,
wherein the Type II anti-CD20 antibody is an IgG antibody,
particularly an IgG.sub.1 antibody.
[0702] 8. The method of any one of the preceding embodiments,
wherein the Type II anti-CD20 antibody is engineered to have an
increased proportion of non-fucosylated oligosaccharides in the Fc
region as compared to a non-engineered antibody.
[0703] 9. The method of any one of the preceding embodiments,
wherein at least about 40% of the N-linked oligosaccharides in the
Fc region of the Type II anti-CD20 antibody are
non-fucosylated.
[0704] 10. The method of any one of the preceding embodiments,
wherein the Type II anti-CD20 antibody is obinutuzumab.
[0705] 11. The method of any one of the preceding embodiments,
wherein the therapeutic agent comprises a polypeptide.
[0706] 12. The method of any one of the preceding embodiments,
wherein the therapeutic agent comprises an antibody.
[0707] 13. The method of embodiment 12, wherein the antibody
comprised in the therapeutic agent specifically binds to
carcinoembryonic antigen (CEA).
[0708] 14. The method of embodiment 13, wherein the antibody
comprised in the therapeutic agent comprises a heavy chain variable
region comprising the heavy chain CDR (HCDR) 1 of SEQ ID NO: 14,
the HCDR2 of SEQ ID NO: 15, and the HCDR3 of SEQ ID NO: 16; and a
light chain variable region comprising the light chain CDR (LCDR) 1
of SEQ ID NO: 17, the LCDR2 of SEQ ID NO: 18 and the LCDR3 of SEQ
ID NO: 19.
[0709] 15. The method of embodiment 13 or 14, wherein the antibody
comprised in the therapeutic agent comprises the heavy chain
variable region sequence of SEQ ID NO: 20 and the light chain
variable region sequence of SEQ ID NO: 21.
[0710] 16. The method of embodiment 12, wherein the antibody
comprised in the therapeutic agent specifically binds to CD3,
particularly CD3.epsilon..
[0711] 17. The method of embodiment 16, wherein the antibody
comprised in the therapeutic agent comprises a heavy chain variable
region comprising the heavy chain CDR (HCDR) 1 of SEQ ID NO: 32,
the HCDR2 of SEQ ID NO: 33, and the HCDR3 of SEQ ID NO: 34; and a
light chain variable region comprising the light chain CDR (LCDR) 1
of SEQ ID NO: 35, the LCDR2 of SEQ ID NO: 36 and the LCDR3 of SEQ
ID NO: 37.
[0712] 18. The method of embodiment 16 or 17, wherein the antibody
comprised in the therapeutic agent comprises the heavy chain
variable region sequence of SEQ ID NO: 38 and the light chain
variable region sequence of SEQ ID NO: 39.
[0713] 19. The method of any one of embodiments 1 to 18, wherein
the therapeutic agent comprises a cytokine.
[0714] 20. The method of embodiment 19, wherein the cytokine is
interleukin-2 (IL-2).
[0715] 21. The method of embodiment 19 or 20, wherein the cytokine
is a mutant human IL-2 polypeptide comprising the amino acid
substitutions F42A, Y45A and L72G (numbering relative to the human
IL-2 sequence SEQ ID NO: 12).
[0716] 22. The method of any one of the preceding embodiments,
wherein the therapeutic agent comprises an immunoconjugate.
[0717] 23. The method of embodiment 22, wherein the immunoconjugate
comprises an antibody as defined in any one of embodiments 13 to
15, and a cytokine as defined in embodiment 20 or 21.
[0718] 24. The method of any one of the preceding embodiments,
wherein the therapeutic agent comprises cergutuzumab amunaleukin
(CEA-IL2v).
[0719] 25. The method of any one of embodiments 1 to 18, wherein
the therapeutic agent comprises a bispecific antibody comprising an
antibody as defined in any one of embodiments 13 to 15 and an
antibody as defined in any one of embodiments 16 to 18.
[0720] 26. A Type II anti-CD20 antibody for use in a method of
treating a disease in a subject, the method comprising a treatment
regimen comprising
[0721] (i) administration to the subject of the Type II anti-CD20
antibody,
[0722] and consecutively after a period of time
[0723] (ii) administration to the subject of a therapeutic
agent,
[0724] wherein the period of time between the administration of the
Type II anti-CD20 antibody and the administration of the
therapeutic agent is sufficient for reduction of the number of
B-cells in the subject in response to the administration of the
Type II anti-CD20 antibody.
[0725] 27. The Type II anti-CD20 antibody of embodiment 26, wherein
the treatment regimen effectively reduces the formation of
anti-drug antibodies (ADAs) in the subject in response to the
administration of the therapeutic agent as compared to a
corresponding treatment regimen without the administration of the
Type II anti-CD20 antibody.
[0726] 28. A Type II anti-CD20 antibody for use in a method for
reducing the formation of anti-drug antibodies (ADAs) against a
therapeutic agent in a subject, comprising administration of the
Type II anti-CD20 antibody to the subject prior to administration
of the therapeutic agent.
[0727] 29. The Type II anti-CD20 antibody of embodiment 28, wherein
the period of time between the administration of the Type II
anti-CD20 antibody and administration of the therapeutic agent is
sufficient for reduction of the number of B-cells in the subject in
response to the administration of the Type II anti-CD20
antibody.
[0728] 30. The Type II anti-CD20 antibody of any one of embodiments
26 to 29, wherein the Type II anti-CD20 antibody comprises a heavy
chain variable region comprising the heavy chain CDR (HCDR) 1 of
SEQ ID NO: 4, the HCDR2 of SEQ ID NO: 5, and the HCDR3 of SEQ ID
NO: 6; and a light chain variable region comprising the light chain
CDR (LCDR) 1 of SEQ ID NO: 7, the LCDR2 of SEQ ID NO: 8 and the
LCDR3 of SEQ ID NO: 9.
[0729] 31. The Type II anti-CD20 antibody of any one of embodiments
26 to 30, wherein the Type II anti-CD20 antibody comprises the
heavy chain variable region sequence of SEQ ID NO: 10 and the light
chain variable region sequence of SEQ ID NO: 11.
[0730] 32. The Type II anti-CD20 antibody of any one of embodiments
26 to 31, wherein the Type II anti-CD20 antibody is an IgG
antibody, particularly an IgG.sub.1 antibody.
[0731] 33. The Type II anti-CD20 antibody of any one of embodiments
26 to 32, wherein the Type II anti-CD20 antibody is engineered to
have an increased proportion of non-fucosylated oligosaccharides in
the Fc region as compared to a non-engineered antibody.
[0732] 34. The Type II anti-CD20 antibody of any one of embodiments
26 to 33, wherein at least about 40% of the N-linked
oligosaccharides in the Fc region of the anti-CD20 antibody are
non-fucosylated.
[0733] 35. The Type II anti-CD20 antibody of any one of embodiments
26 to 34, wherein the Type II anti-CD20 antibody is
obinutuzumab.
[0734] 36. The Type II anti-CD20 antibody of any one of embodiments
26 to 35, wherein the therapeutic agent comprises a
polypeptide.
[0735] 37. The Type II anti-CD20 antibody of any one of embodiments
26 to 36, wherein the therapeutic agent comprises an antibody.
[0736] 38. The Type II anti-CD20 antibody of embodiment 37, wherein
the antibody comprised in the therapeutic agent specifically binds
to carcinoembryonic antigen (CEA).
[0737] 39. The Type II anti-CD20 antibody of embodiment 38, wherein
the antibody comprised in the therapeutic agent comprises a heavy
chain variable region comprising the heavy chain CDR (HCDR) 1 of
SEQ ID NO: 14, the HCDR2 of SEQ ID NO: 15, and the HCDR3 of SEQ ID
NO: 16; and a light chain variable region comprising the light
chain CDR (LCDR) 1 of SEQ ID NO: 17, the LCDR2 of SEQ ID NO: 18 and
the LCDR3 of SEQ ID NO: 19.
[0738] 40. The Type II anti-CD20 antibody of embodiment 38 or 39,
wherein the antibody comprised in the therapeutic agent comprises
the heavy chain variable region sequence of SEQ ID NO: 20 and the
light chain variable region sequence of SEQ ID NO: 21.
[0739] 41. The Type II anti-CD20 antibody of embodiment 37, wherein
the antibody comprised in the therapeutic agent specifically binds
to CD3, particularly CD3.epsilon..
[0740] 42. The Type II anti-CD20 antibody of embodiment 41, wherein
the antibody comprised in the therapeutic agent comprises a heavy
chain variable region comprising the heavy chain CDR (HCDR) 1 of
SEQ ID NO: 32, the HCDR2 of SEQ ID NO: 33, and the HCDR3 of SEQ ID
NO: 34; and a light chain variable region comprising the light
chain CDR (LCDR) 1 of SEQ ID NO: 35, the LCDR2 of SEQ ID NO: 36 and
the LCDR3 of SEQ ID NO: 37.
[0741] 43. The Type II anti-CD20 antibody of embodiment 41 or 42,
wherein the antibody comprised in the therapeutic agent comprises
the heavy chain variable region sequence of SEQ ID NO: 38 and the
light chain variable region sequence of SEQ ID NO: 39.
[0742] 44. The Type II anti-CD20 antibody of any one of embodiments
26 to 43, wherein the therapeutic agent comprises a cytokine.
[0743] 45. The Type II anti-CD20 antibody of embodiment 44, wherein
the cytokine is interleukin-2 (IL-2).
[0744] 46. The Type II anti-CD20 antibody of embodiment 44 or 45,
wherein the cytokine is a mutant human IL-2 polypeptide comprising
the amino acid substitutions F42A, Y45A and L72G (numbering
relative to the human IL-2 sequence SEQ ID NO: 12).
[0745] 47. The Type II anti-CD20 antibody of any one of embodiments
26 to 46, wherein the therapeutic agent comprises an
immunoconjugate.
[0746] 48. The Type II anti-CD20 antibody of embodiment 47, wherein
the immunoconjugate comprises an antibody as defined in any one of
embodiments 38 to 40, and a cytokine as defined in embodiment 45 or
46.
[0747] 49. The Type II anti-CD20 antibody of any one of embodiments
26 to 48, wherein the therapeutic agent comprises cergutuzumab
amunaleukin (CEA-IL2v).
[0748] 50. The Type II anti-CD20 antibody of any one of embodiments
1 to 43, wherein the therapeutic agent comprises a bispecific
antibody comprising an antibody as defined in any one of
embodiments 38 to 40 and an antibody as defined in any one of
embodiments 41 to 43.
[0749] 51. Use of a Type II anti-CD20 antibody in the manufacture
of a medicament for reduction of the formation of anti-drug
antibodies (ADAs) against a therapeutic agent in a subject,
[0750] wherein the medicament is to be used in a treatment regimen
comprising
[0751] (i) administration to the subject of the Type II anti-CD20
antibody,
[0752] and consecutively after a period of time
[0753] (ii) administration to the subject of a therapeutic
agent,
[0754] wherein the period of time between the administration of the
Type II anti-CD20 antibody and the administration of the
therapeutic agent is sufficient for reduction of the number of
B-cells in the subject in response to the administration of the
Type II anti-CD20 antibody.
[0755] 52. Use of a therapeutic agent in the manufacture of a
medicament for treatment of a disease in a subject, wherein the
treatment comprises a treatment regimen comprising
[0756] (i) administration to the subject of a Type II anti-CD20
antibody,
[0757] and consecutively after a period of time
[0758] (ii) administration to the subject of the therapeutic
agent,
[0759] wherein the period of time between the administration of the
Type II anti-CD20 antibody and the administration of the
therapeutic agent is sufficient for reduction of the number of
B-cells in the subject in response to the administration of the
Type II anti-CD20 antibody.
[0760] 53. The use of embodiment 51 or 52, wherein the treatment
regimen effectively reduces the formation of anti-drug antibodies
(ADAs) against the therapeutic agent in the subject as compared to
a corresponding treatment regimen without the administration of the
Type II anti-CD20 antibody.
[0761] 54. The use of any one of embodiments 51 to 53, wherein the
Type II anti-CD20 antibody comprises a heavy chain variable region
comprising the heavy chain CDR (HCDR) 1 of SEQ ID NO: 4, the HCDR2
of SEQ ID NO: 5, and the HCDR3 of SEQ ID NO: 6; and a light chain
variable region comprising the light chain CDR (LCDR) 1 of SEQ ID
NO: 7, the LCDR2 of SEQ ID NO: 8 and the LCDR3 of SEQ ID NO: 9.
[0762] 55. The use of any one of embodiments 51 to 54, wherein the
Type II anti-CD20 antibody comprises the heavy chain variable
region sequence of SEQ ID NO: 10 and the light chain variable
region sequence of SEQ ID NO: 11.
[0763] 56. The use of any one of embodiments 51 to 55, wherein the
Type II anti-CD20 antibody is an IgG antibody, particularly an
IgG.sub.1 antibody.
[0764] 57. The use of any one of embodiments 51 to 56, wherein the
Type II anti-CD20 antibody is engineered to have an increased
proportion of non-fucosylated oligosaccharides in the Fc region as
compared to a non-engineered antibody.
[0765] 58. The use of any one of embodiments 51 to 57, wherein at
least about 40% of the N-linked oligosaccharides in the Fc region
of the Type II anti-CD20 antibody are non-fucosylated.
[0766] 59. The use of any one of embodiments 51 to 58, wherein the
Type II anti-CD20 antibody is obinutuzumab.
[0767] 60. The use of any one of embodiments 51 to 59, wherein the
therapeutic agent comprises a polypeptide.
[0768] 61. The use of any one of embodiments 51 to 60, wherein the
therapeutic agent comprises an antibody.
[0769] 62. The use of embodiment 61, wherein the antibody comprised
in the therapeutic agent specifically binds to carcinoembryonic
antigen (CEA).
[0770] 63. The use of embodiment 62, wherein the antibody comprised
in the therapeutic agent comprises a heavy chain variable region
comprising the heavy chain CDR (HCDR) 1 of SEQ ID NO: 14, the HCDR2
of SEQ ID NO: 15, and the HCDR3 of SEQ ID NO: 16; and a light chain
variable region comprising the light chain CDR (LCDR) 1 of SEQ ID
NO: 17, the LCDR2 of SEQ ID NO: 18 and the LCDR3 of SEQ ID NO:
19.
[0771] 64. The use of embodiment 62 or 63, wherein the antibody
comprised in the therapeutic agent comprises the heavy chain
variable region sequence of SEQ ID NO: 20 and the light chain
variable region sequence of SEQ ID NO: 21.
[0772] 65. The use of embodiment 61, wherein the antibody comprised
in the therapeutic agent specifically binds to CD3, particularly
CD3.epsilon..
[0773] 66. The use of embodiment 65, wherein the antibody comprised
in the therapeutic agent comprises a heavy chain variable region
comprising the heavy chain CDR (HCDR) 1 of SEQ ID NO: 32, the HCDR2
of SEQ ID NO: 33, and the HCDR3 of SEQ ID NO: 34; and a light chain
variable region comprising the light chain CDR (LCDR) 1 of SEQ ID
NO: 35, the LCDR2 of SEQ ID NO: 36 and the LCDR3 of SEQ ID NO:
37.
[0774] 67. The use of embodiment 65 or 66, wherein the antibody
comprised in the therapeutic agent comprises the heavy chain
variable region sequence of SEQ ID NO: 38 and the light chain
variable region sequence of SEQ ID NO: 39.
[0775] 68. The use of any one of embodiments 51 to 67, wherein the
therapeutic agent comprises a cytokine.
[0776] 69. The use of embodiment 68, wherein the cytokine is
interleukin-2 (IL-2).
[0777] 70. The use of embodiment 68 or 69, wherein the cytokine is
a mutant human IL-2 polypeptide comprising the amino acid
substitutions F42A, Y45A and L72G (numbering relative to the human
IL-2 sequence SEQ ID NO: 12).
[0778] 71. The use of any one of embodiments 51 to 70, wherein the
therapeutic agent comprises an immunoconjugate.
[0779] 72. The use of embodiment 71, wherein the immunoconjugate
comprises an antibody as defined in any one of embodiments 62 to
64, and a cytokine as defined in embodiment 69 or 70.
[0780] 73. The use of any one of embodiments 51 to 72, wherein the
therapeutic agent comprises cergutuzumab amunaleukin
(CEA-IL2v).
[0781] 74. The use of any one of embodiments 51 to 67, wherein the
therapeutic agent comprises a bispecific antibody comprising an
antibody as defined in any one of embodiments 62 to 64 and an
antibody as defined in any one of embodiments 65 to 67.
[0782] 75. A kit for the reduction of the formation of anti-drug
antibodies (ADAs) against a therapeutic agent in a subject,
comprising a package comprising a Type II anti-CD20 antibody
composition and instructions for using the Type II anti-CD20
antibody composition in a treatment regimen comprising
[0783] (iii) administration to the subject of the Type II anti-CD20
antibody composition,
[0784] and consecutively after a period of time
[0785] (iv) administration to the subject of a therapeutic
agent,
[0786] wherein the period of time between the administration of the
Type II anti-CD20 antibody composition and the administration of
the therapeutic agent is sufficient for reduction of the number of
B-cells in the subject in response to the administration of the
Type II anti-CD20 antibody.
[0787] 76. The kit of embodiment 75, further comprising a
therapeutic agent composition.
[0788] 77. A kit for the treatment of a disease in a subject,
comprising a package comprising a therapeutic agent composition and
instructions for using the therapeutic agent composition in a
treatment regimen comprising
[0789] (iii) administration to the subject of a Type II anti-CD20
antibody,
[0790] and consecutively after a period of time
[0791] (iv) administration to the subject of the therapeutic agent
composition,
[0792] wherein the period of time between the administration of the
Type II anti-CD20 antibody and the administration of the
therapeutic agent composition is sufficient for reduction of the
number of B-cells in the subject in response to the administration
of the Type II anti-CD20 antibody.
[0793] 78. The kit of embodiment 77, further comprising a Type II
anti-CD20 antibody composition.
[0794] 79. The kit of any one of embodiments 75 to 78, wherein the
treatment regimen effectively reduces the formation of anti-drug
antibodies (ADAs) against the therapeutic agent in the subject as
compared to a corresponding treatment regimen without the
administration of the Type II anti-CD20 antibody composition.
[0795] 80. The kit of any one of embodiments 75 to 79, wherein the
Type II anti-CD20 antibody comprises a heavy chain variable region
comprising the heavy chain CDR (HCDR) 1 of SEQ ID NO: 4, the HCDR2
of SEQ ID NO: 5, and the HCDR3 of SEQ ID NO: 6; and a light chain
variable region comprising the light chain CDR (LCDR) 1 of SEQ ID
NO: 7, the LCDR2 of SEQ ID NO: 8 and the LCDR3 of SEQ ID NO: 9.
[0796] 81. The kit of any one of embodiments 75 to 80, wherein the
Type II anti-CD20 antibody comprises the heavy chain variable
region sequence of SEQ ID NO: 10 and the light chain variable
region sequence of SEQ ID NO: 11.
[0797] 82. The kit of any one of embodiments 75 to 81, wherein the
Type II anti-CD20 antibody is an IgG antibody, particularly an
IgG.sub.1 antibody.
[0798] 83. The kit of any one of embodiments 75 to 82, wherein the
Type II anti-CD20 antibody is engineered to have an increased
proportion of non-fucosylated oligosaccharides in the Fc region as
compared to a non-engineered antibody.
[0799] 84. The kit of any one of embodiments 75 to 83, wherein at
least about 40% of the N-linked oligosaccharides in the Fc region
of the Type II anti-CD20 antibody are non-fucosylated.
[0800] 85. The kit of any one of embodiments 75 to 84, wherein the
Type II anti-CD20 antibody is obinutuzumab.
[0801] 86. The kit of any one of embodiments 75 to 85, wherein the
therapeutic agent comprises a polypeptide.
[0802] 87. The kit of any one of embodiments 75 to 86, wherein the
therapeutic agent comprises an antibody.
[0803] 88. The kit of embodiment 87, wherein the antibody comprised
in the therapeutic agent specifically binds to carcinoembryonic
antigen (CEA).
[0804] 89. The kit of embodiment 88, wherein the antibody comprised
in the therapeutic agent comprises a heavy chain variable region
comprising the heavy chain CDR (HCDR) 1 of SEQ ID NO: 14, the HCDR2
of SEQ ID NO: 15, and the HCDR3 of SEQ ID NO: 16; and a light chain
variable region comprising the light chain CDR (LCDR) 1 of SEQ ID
NO: 17, the LCDR2 of SEQ ID NO: 18 and the LCDR3 of SEQ ID NO:
19.
[0805] 90. The kit of embodiment 88 or 89, wherein the antibody
comprised in the therapeutic agent comprises the heavy chain
variable region sequence of SEQ ID NO: 20 and the light chain
variable region sequence of SEQ ID NO: 21.
[0806] 91. The kit of embodiment 87, wherein the antibody comprised
in the therapeutic agent specifically binds to CD3, particularly
CD3.epsilon..
[0807] 92. The kit of embodiment 91, wherein the antibody comprised
in the therapeutic agent comprises a heavy chain variable region
comprising the heavy chain CDR (HCDR) 1 of SEQ ID NO: 32, the HCDR2
of SEQ ID NO: 33, and the HCDR3 of SEQ ID NO: 34; and a light chain
variable region comprising the light chain CDR (LCDR) 1 of SEQ ID
NO: 35, the LCDR2 of SEQ ID NO: 36 and the LCDR3 of SEQ ID NO:
37.
[0808] 93. The kit of embodiment 91 or 92, wherein the antibody
comprised in the therapeutic agent comprises the heavy chain
variable region sequence of SEQ ID NO: 38 and the light chain
variable region sequence of SEQ ID NO: 39.
[0809] 94. The kit of any one of embodiments 75 to 93, wherein the
therapeutic agent comprises a cytokine.
[0810] 95. The kit of embodiment 94, wherein the cytokine is
interleukin-2 (IL-2).
[0811] 96. The kit of embodiment 94 or 95, wherein the cytokine is
a mutant human IL-2 polypeptide comprising the amino acid
substitutions F42A, Y45A and L72G (numbering relative to the human
IL-2 sequence SEQ ID NO: 12).
[0812] 97. The kit of any one of embodiments 75 to 96, wherein the
therapeutic agent comprises an immunoconjugate.
[0813] 98. The kit of embodiment 97, wherein the immunoconjugate
comprises an antibody as defined in any one of embodiments 88 to
90, and a cytokine as defined in embodiment 95 or 96.
[0814] 99. The kit of any one of embodiments 75 to 98, wherein the
therapeutic agent comprises cergutuzumab amunaleukin
(CEA-IL2v).
[0815] 100. The kit of any one of embodiments 75 to 93, wherein the
therapeutic agent comprises a bispecific antibody comprising an
antibody as defined in any one of embodiments 88 to 90 and an
antibody as defined in any one of embodiments 91 to 93.
[0816] 101. A therapeutic agent for use in a method of treating a
disease in a subject, the method comprising a treatment regimen
comprising
[0817] (i) administration to the subject of a Type II anti-CD20
antibody,
[0818] and consecutively after a period of time
[0819] (ii) administration to the subject of the therapeutic
agent,
[0820] wherein the period of time between the administration of the
Type II anti-CD20 antibody and the administration of the
therapeutic agent is sufficient for reduction of the number of
B-cells in the subject in response to the administration of the
Type II anti-CD20 antibody.
[0821] 102. The therapeutic agent of embodiment 101, wherein the
treatment regimen effectively reduces the formation of anti-drug
antibodies (ADAs) in the subject in response to the administration
of the therapeutic agent as compared to a corresponding treatment
regimen without the administration of the Type II anti-CD20
antibody.
[0822] 103. The therapeutic agent of embodiment 101 or 102, wherein
the Type II anti-CD20 antibody comprises a heavy chain variable
region comprising the heavy chain CDR (HCDR) 1 of SEQ ID NO: 4, the
HCDR2 of SEQ ID NO: 5, and the HCDR3 of SEQ ID NO: 6; and a light
chain variable region comprising the light chain CDR (LCDR) 1 of
SEQ ID NO: 7, the LCDR2 of SEQ ID NO: 8 and the LCDR3 of SEQ ID NO:
9.
[0823] 104. The therapeutic agent of any one of embodiments 101 to
103, wherein the Type II anti-CD20 antibody comprises the heavy
chain variable region sequence of SEQ ID NO: 10 and the light chain
variable region sequence of SEQ ID NO: 11.
[0824] 105. The therapeutic agent of any one of embodiments 101 to
104, wherein the Type II anti-CD20 antibody is an IgG antibody,
particularly an IgG.sub.1 antibody.
[0825] 106. The therapeutic agent of any one of embodiments 101 to
105, wherein the Type II anti-CD20 antibody is engineered to have
an increased proportion of non-fucosylated oligosaccharides in the
Fc region as compared to a non-engineered antibody.
[0826] 107. The therapeutic agent of any one of embodiments 101 to
106, wherein at least about 40% of the N-linked oligosaccharides in
the Fc region of the Type II anti-CD20 antibody are
non-fucosylated.
[0827] 108. The therapeutic agent of any one of embodiments 101 to
107, wherein the Type II anti-CD20 antibody is obinutuzumab.
[0828] 109. The therapeutic agent of any one of embodiments 101 to
108, wherein the therapeutic agent comprises a polypeptide.
[0829] 110. The therapeutic agent of any one of embodiments 101 to
109, wherein the therapeutic agent comprises an antibody.
[0830] 111. The therapeutic agent of embodiment 110, wherein the
antibody comprised in the therapeutic agent specifically binds to
carcinoembryonic antigen (CEA).
[0831] 112. The therapeutic agent of embodiment 111, wherein the
antibody comprised in the therapeutic agent comprises a heavy chain
variable region comprising the heavy chain CDR (HCDR) 1 of SEQ ID
NO: 14, the HCDR2 of SEQ ID NO: 15, and the HCDR3 of SEQ ID NO: 16;
and a light chain variable region comprising the light chain CDR
(LCDR) 1 of SEQ ID NO: 17, the LCDR2 of SEQ ID NO: 18 and the LCDR3
of SEQ ID NO: 19.
[0832] 113. The therapeutic agent of embodiment 110 or 111, wherein
the antibody comprised in the therapeutic agent comprises the heavy
chain variable region sequence of SEQ ID NO: 20 and the light chain
variable region sequence of SEQ ID NO: 21.
[0833] 114. The therapeutic agent of embodiment 110, wherein the
antibody comprised in the therapeutic agent specifically binds to
CD3, particularly CD3.epsilon..
[0834] 115. The therapeutic agent of embodiment 114, wherein the
antibody comprised in the therapeutic agent comprises a heavy chain
variable region comprising the heavy chain CDR (HCDR) 1 of SEQ ID
NO: 32, the HCDR2 of SEQ ID NO: 33, and the HCDR3 of SEQ ID NO: 34;
and a light chain variable region comprising the light chain CDR
(LCDR) 1 of SEQ ID NO: 35, the LCDR2 of SEQ ID NO: 36 and the LCDR3
of SEQ ID NO: 37.
[0835] 116. The therapeutic agent of embodiment 114 or 115, wherein
the antibody comprised in the therapeutic agent comprises the heavy
chain variable region sequence of SEQ ID NO: 38 and the light chain
variable region sequence of SEQ ID NO: 39.
[0836] 117. The therapeutic agent of any one of embodiments 101 to
116, wherein the therapeutic agent comprises a cytokine.
[0837] 118. The therapeutic agent of embodiment 117, wherein the
cytokine is interleukin-2 (IL-2).
[0838] 119. The therapeutic agent of embodiment 117 or 118, wherein
the cytokine is a mutant human IL-2 polypeptide comprising the
amino acid substitutions F42A, Y45A and L72G (numbering relative to
the human IL-2 sequence SEQ ID NO: 12).
[0839] 120. The therapeutic agent of any one of embodiments 101 to
119, wherein the therapeutic agent comprises an
immunoconjugate.
[0840] 121. The therapeutic agent of embodiment 120, wherein the
immunoconjugate comprises an antibody as defined in any one of
embodiments 111 to 113, and a cytokine as defined in embodiment 118
or 119.
[0841] 122. The therapeutic agent of any one of embodiments 101 to
121, wherein the therapeutic agent comprises cergutuzumab
amunaleukin (CEA-IL2v).
[0842] 123. The therapeutic agent of any one of embodiments 101 to
119, wherein the therapeutic agent comprises a bispecific antibody
comprising an antibody as defined in any one of embodiments 111 to
113 and an antibody as defined in any one of embodiments 114 to
116.
[0843] In the following, further embodiments of the invention are
listed.
[0844] 1. A method of treating a disease in a subject, the method
comprising a treatment regimen comprising
[0845] (i) administration to the subject of a Type II anti-CD20
antibody,
[0846] and consecutively after a period of time
[0847] (ii) administration to the subject of a T-cell activating
therapeutic agent,
[0848] wherein the period of time between the administration of the
Type II anti-CD20 antibody and the administration of the
therapeutic agent is sufficient for reduction of the number of
B-cells in the subject in response to the administration of the
CD20 antibody.
[0849] 2. The method of embodiment 1, wherein the treatment regimen
effectively reduces cytokine release in the subject associated with
the administration of the therapeutic agent as compared to a
corresponding treatment regimen without the administration of the
Type II anti-CD20 antibody.
[0850] 3. A method for reducing cytokine release associated with
the administration of a therapeutic agent in a subject, comprising
administration of a Type II anti-CD20 antibody to the subject prior
to administration of the therapeutic agent.
[0851] 4. The method of embodiment 3, wherein the period of time
between the administration of the Type II anti-CD20 antibody and
administration of the therapeutic agent is sufficient for reduction
of the number of B-cells in the subject in response to the
administration of the Type II anti-CD20 antibody.
[0852] 5. The method of any one of the preceding embodiments,
wherein the Type II anti-CD20 antibody comprises a heavy chain
variable region comprising the heavy chain CDR (HCDR) 1 of SEQ ID
NO: 4, the HCDR2 of SEQ ID NO: 5, and the HCDR3 of SEQ ID NO: 6;
and a light chain variable region comprising the light chain CDR
(LCDR) 1 of SEQ ID NO: 7, the LCDR2 of SEQ ID NO: 8 and the LCDR3
of SEQ ID NO: 9.
[0853] 6. The method of any one of the preceding embodiments,
wherein the Type II anti-CD20 antibody comprises the heavy chain
variable region sequence of SEQ ID NO: 10 and the light chain
variable region sequence of SEQ ID NO: 11.
[0854] 7. The method of any one of the preceding embodiments,
wherein the Type II anti-CD20 antibody is an IgG antibody,
particularly an IgG.sub.1 antibody.
[0855] 8. The method of any one of the preceding embodiments,
wherein the Type II anti-CD20 antibody is engineered to have an
increased proportion of non-fucosylated oligosaccharides in the Fc
region as compared to a non-engineered antibody.
[0856] 9. The method of any one of the preceding embodiments,
wherein at least about 40% of the N-linked oligosaccharides in the
Fc region of the Type II anti-CD20 antibody are
non-fucosylated.
[0857] 10. The method of any one of the preceding embodiments,
wherein the Type II anti-CD20 antibody is obinutuzumab.
[0858] 11. The method of any one of the preceding embodiments,
wherein the therapeutic agent comprises an antibody, particularly a
multispecific antibody.
[0859] 12. The method of embodiment 11, wherein the antibody
comprised in the therapeutic agent specifically binds to an
activating T cell antigen, particularly an antigen selected from
the group consisting of CD3, CD28, CD137 (also known as 4-1BB),
CD40, CD226, OX40, GITR, CD27, HVEM, and CD127, more particularly
CD3, most particularly CD3.epsilon..
[0860] 13. The method of embodiment 11 or 12, wherein the antibody
comprised in the therapeutic agent comprises a heavy chain variable
region comprising the heavy chain CDR (HCDR) 1 of SEQ ID NO: 32,
the HCDR2 of SEQ ID NO: 33, and the HCDR3 of SEQ ID NO: 34; and a
light chain variable region comprising the light chain CDR (LCDR) 1
of SEQ ID NO: 35, the LCDR2 of SEQ ID NO: 36 and the LCDR3 of SEQ
ID NO: 37.
[0861] 14. The method of any one of embodiments 11 to 13, wherein
the antibody comprised in the therapeutic agent comprises the heavy
chain variable region sequence of SEQ ID NO: 38 and the light chain
variable region sequence of SEQ ID NO: 39.
[0862] 15. The method of any one of embodiments 11 to 14, wherein
the antibody comprised in the therapeutic agent specifically binds
to a B-cell antigen, particularly an antigen selected from the
group consisting of CD20, CD19, CD22, ROR-1, CD37 and CD5, more
particularly CD20 or CD19, most particularly CD20.
[0863] 16. The method of embodiment 15, wherein the antibody
comprised in the therapeutic agent comprises a heavy chain variable
region comprising the heavy chain CDR (HCDR) 1 of SEQ ID NO: 4, the
HCDR2 of SEQ ID NO: 5, and the HCDR3 of SEQ ID NO: 6; and a light
chain variable region comprising the light chain CDR (LCDR) 1 of
SEQ ID NO: 7, the LCDR2 of SEQ ID NO: 8 and the LCDR3 of SEQ ID NO:
9.
[0864] 17. The method of embodiment 15 or 16, wherein the antibody
comprised in the therapeutic agent comprises the heavy chain
variable region sequence of SEQ ID NO: 10 and the light chain
variable region sequence of SEQ ID NO: 11.
[0865] 18. The method of any one of the preceding embodiments,
wherein the antibody comprised in the therapeutic agent is a
bispecific antibody comprising (i) an antibody as defined in any
one of embodiments 12 to 14 and (ii) an antibody as defined in any
one of embodiments 15 to 17.
[0866] 19. The method of any one of the preceding embodiments,
wherein the therapeutic agent comprises CD20XCD3 bsAB.
[0867] 20. The method of any one of embodiments 1 to 10, wherein
the therapeutic agent comprises a T cell expressing a chimeric
antigen receptor (CAR), particularly a CAR that specifically binds
to a B-cell antigen, more particularly a CAR that specifically
binds to an antigen selected from the group of CD20, CD19, CD22,
ROR-1, CD37 and CD5.
[0868] 21. The method of any one of the preceding embodiments,
wherein the disease is a B cell proliferative disorder,
particularly a CD20-positive B-cell disorder, and/or is a disease
selected from the group consisting of Non-Hodgkin lymphoma (NHL),
acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia
(CLL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma
(FL), mantle-cell lymphoma (MCL), marginal zone lymphoma (MZL),
Multiple myeloma (MM) and Hodgkin lymphoma (HL).
[0869] 22. A Type II anti-CD20 antibody for use in a method of
treating a disease in a subject, the method comprising a treatment
regimen comprising
[0870] (i) administration to the subject of the Type II anti-CD20
antibody,
[0871] and consecutively after a period of time
[0872] (ii) administration to the subject of a T-cell activating
therapeutic agent,
[0873] wherein the period of time between the administration of the
Type II anti-CD20 antibody and the administration of the
therapeutic agent is sufficient for reduction of the number of
B-cells in the subject in response to the administration of the
Type II anti-CD20 antibody.
[0874] 23. The Type II anti-CD20 antibody of embodiment 22, wherein
the treatment regimen effectively reduces cytokine release in the
subject associated with the administration of the therapeutic agent
as compared to a corresponding treatment regimen without the
administration of the Type II anti-CD20 antibody.
[0875] 24. A Type II anti-CD20 antibody for use in a method for
reducing cytokine release associated with the administration of a
therapeutic agent in a subject, comprising administration of the
Type II anti-CD20 antibody to the subject prior to administration
of the therapeutic agent.
[0876] 25. The Type II anti-CD20 antibody of embodiment 24, wherein
the period of time between the administration of the Type II
anti-CD20 antibody and administration of the therapeutic agent is
sufficient for reduction of the number of B-cells in the subject in
response to the administration of the Type II anti-CD20
antibody.
[0877] 26. The Type II anti-CD20 antibody of any one of embodiments
22 to 25, wherein the Type II anti-CD20 antibody comprises a heavy
chain variable region comprising the heavy chain CDR (HCDR) 1 of
SEQ ID NO: 4, the HCDR2 of SEQ ID NO: 5, and the HCDR3 of SEQ ID
NO: 6; and a light chain variable region comprising the light chain
CDR (LCDR) 1 of SEQ ID NO: 7, the LCDR2 of SEQ ID NO: 8 and the
LCDR3 of SEQ ID NO: 9.
[0878] 27. The Type II anti-CD20 antibody of any one of embodiments
22 to 26, wherein the Type II anti-CD20 antibody comprises the
heavy chain variable region sequence of SEQ ID NO: 10 and the light
chain variable region sequence of SEQ ID NO: 11.
[0879] 28. The Type II anti-CD20 antibody of any one of embodiments
22 to 27, wherein the Type II anti-CD20 antibody is an IgG
antibody, particularly an IgG.sub.1 antibody.
[0880] 29. The Type II anti-CD20 antibody of any one of embodiments
22 to 28, wherein the Type II anti-CD20 antibody is engineered to
have an increased proportion of non-fucosylated oligosaccharides in
the Fc region as compared to a non-engineered antibody.
[0881] 30. The Type II anti-CD20 antibody of any one of embodiments
22 to 29, wherein at least about 40% of the N-linked
oligosaccharides in the Fc region of the Type II anti-CD20 antibody
are non-fucosylated.
[0882] 31. The Type II anti-CD20 antibody of any one of embodiments
22 to 30, wherein the Type II anti-CD20 antibody is
obinutuzumab.
[0883] 32. The Type II anti-CD20 antibody of any one of embodiments
22 to 31, wherein the therapeutic agent comprises an antibody,
particularly a multispecific antibody.
[0884] 33. The Type II anti-CD20 antibody of embodiment 32, wherein
the antibody comprised in the therapeutic agent specifically binds
to an activating T cell antigen, particularly an antigen selected
from the group consisting of CD3, CD28, CD137 (also known as
4-1BB), CD40, CD226, OX40, GITR, CD27, HVEM, and CD127, more
particularly CD3, most particularly CD3.epsilon..
[0885] 34. The Type II anti-CD20 antibody of embodiment 32 or 33,
wherein the antibody comprised in the therapeutic agent comprises a
heavy chain variable region comprising the heavy chain CDR (HCDR) 1
of SEQ ID NO: 32, the HCDR2 of SEQ ID NO: 33, and the HCDR3 of SEQ
ID NO: 34; and a light chain variable region comprising the light
chain CDR (LCDR) 1 of SEQ ID NO: 35, the LCDR2 of SEQ ID NO: 36 and
the LCDR3 of SEQ ID NO: 37.
[0886] 35. The Type II anti-CD20 antibody of any one of embodiments
32 to 34, wherein the antibody comprised in the therapeutic agent
comprises the heavy chain variable region sequence of SEQ ID NO: 38
and the light chain variable region sequence of SEQ ID NO: 39.
[0887] 36. The Type II anti-CD20 antibody of any one of embodiments
32 to 35, wherein the antibody comprised in the therapeutic agent
specifically binds to a B-cell antigen, particularly an antigen
selected from the group consisting of CD20, CD19, CD22, ROR-1, CD37
and CD5, more particularly CD20 or CD19, most particularly
CD20.
[0888] 37. The Type II anti-CD20 antibody of embodiment 36, wherein
the antibody comprised in the therapeutic agent comprises a heavy
chain variable region comprising the heavy chain CDR (HCDR) 1 of
SEQ ID NO: 4, the HCDR2 of SEQ ID NO: 5, and the HCDR3 of SEQ ID
NO: 6; and a light chain variable region comprising the light chain
CDR (LCDR) 1 of SEQ ID NO: 7, the LCDR2 of SEQ ID NO: 8 and the
LCDR3 of SEQ ID NO: 9.
[0889] 38. The Type II anti-CD20 antibody of embodiment 36 or 37,
wherein the antibody comprised in the therapeutic agent comprises
the heavy chain variable region sequence of SEQ ID NO: 10 and the
light chain variable region sequence of SEQ ID NO: 11.
[0890] 39. The Type II anti-CD20 antibody of any one of embodiments
22 to 38, wherein the antibody comprised in the therapeutic agent
is a bispecific antibody comprising (i) an antibody as defined in
any one of embodiments 33 to 35 and (ii) an antibody as defined in
any one of embodiments 36 to 38.
[0891] 40. The Type II anti-CD20 antibody of any one of embodiments
22 to 39, wherein the therapeutic agent comprises CD20XCD3
bsAB.
[0892] 41. The Type II anti-CD20 antibody of any one of embodiments
22 to 31, wherein the therapeutic agent comprises a T cell
expressing a chimeric antigen receptor (CAR), particularly a CAR
that specifically binds to a B-cell antigen, more particularly a
CAR that specifically binds to an antigen selected from the group
of CD20, CD19, CD22, ROR-1, CD37 and CD5.
[0893] 42. The Type II anti-CD20 antibody of any one of embodiments
22 to 41, wherein the disease is a B cell proliferative disorder,
particularly a CD20-positive B-cell disorder, and/or is a disease
selected from the group consisting of Non-Hodgkin lymphoma (NHL),
acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia
(CLL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma
(FL), mantle-cell lymphoma (MCL), marginal zone lymphoma (MZL),
Multiple myeloma (MM) and Hodgkin lymphoma (HL).
[0894] 43. Use of a Type II anti-CD20 antibody in the manufacture
of a medicament for reduction of cytokine release associated with
the administration of a T-cell activating therapeutic agent in a
subject, wherein the medicament is to be used in a treatment
regimen comprising
[0895] (i) administration to the subject of the Type II anti-CD20
antibody,
[0896] and consecutively after a period of time
[0897] (ii) administration to the subject of a T-cell activating
therapeutic agent,
[0898] wherein the period of time between the administration of the
Type II anti-CD20 antibody and the administration of the
therapeutic agent is sufficient for reduction of the number of
B-cells in the subject in response to the administration of the
CD20 antibody.
[0899] 44. Use of a T-cell activating therapeutic agent in the
manufacture of a medicament for treatment of a disease in a
subject, wherein the treatment comprises a treatment regimen
comprising
[0900] (iii) administration to the subject of a Type II anti-CD20
antibody,
[0901] and consecutively after a period of time
[0902] (iv) administration to the subject of the T-cell activating
therapeutic agent,
[0903] wherein the period of time between the administration of the
Type II anti-CD20 antibody and the administration of the
therapeutic agent is sufficient for reduction of the number of
B-cells in the subject in response to the administration of the
CD20 antibody.
[0904] 45. The use of embodiment 43 or 44, wherein the treatment
regimen effectively reduces cytokine release associated with the
administration of the therapeutic agent in the subject as compared
to a corresponding treatment regimen without the administration of
the Type II anti-CD20 antibody.
[0905] 46. The use of any one of embodiments 43 to 45, wherein the
Type II anti-CD20 antibody comprises a heavy chain variable region
comprising the heavy chain CDR (HCDR) 1 of SEQ ID NO: 4, the HCDR2
of SEQ ID NO: 5, and the HCDR3 of SEQ ID NO: 6; and a light chain
variable region comprising the light chain CDR (LCDR) 1 of SEQ ID
NO: 7, the LCDR2 of SEQ ID NO: 8 and the LCDR3 of SEQ ID NO: 9.
[0906] 47. The use of any one of embodiments 43 to 46, wherein the
Type II anti-CD20 antibody comprises the heavy chain variable
region sequence of SEQ ID NO: 10 and the light chain variable
region sequence of SEQ ID NO: 11.
[0907] 48. The use of any one of embodiments 43 to 47, wherein the
Type II anti-CD20 antibody is an IgG antibody, particularly an
IgG.sub.1 antibody.
[0908] 49. The use of any one of embodiments 43 to 48, wherein the
Type II anti-CD20 antibody is engineered to have an increased
proportion of non-fucosylated oligosaccharides in the Fc region as
compared to a non-engineered antibody.
[0909] 50. The use of any one of embodiments 43 to 49, wherein at
least about 40% of the N-linked oligosaccharides in the Fc region
of the Type II anti-CD20 antibody are non-fucosylated.
[0910] 51. The use of any one of embodiments 43 to 50, wherein the
Type II anti-CD20 antibody is obinutuzumab.
[0911] 52. The use of any one of embodiments 43 to 51, wherein the
therapeutic agent comprises an antibody, particularly a
multispecific antibody.
[0912] 53. The use of embodiment 51, wherein the antibody comprised
in the therapeutic agent specifically binds to an activating T cell
antigen, particularly an antigen selected from the group consisting
of CD3, CD28, CD137 (also known as 4-1BB), CD40, CD226, OX40, GITR,
CD27, HVEM, and CD127, more particularly CD3, most particularly
CD3.epsilon..
[0913] 54. The use of embodiment 52 or 53, wherein the antibody
comprised in the therapeutic agent comprises a heavy chain variable
region comprising the heavy chain CDR (HCDR) 1 of SEQ ID NO: 32,
the HCDR2 of SEQ ID NO: 33, and the HCDR3 of SEQ ID NO: 34; and a
light chain variable region comprising the light chain CDR (LCDR) 1
of SEQ ID NO: 35, the LCDR2 of SEQ ID NO: 36 and the LCDR3 of SEQ
ID NO: 37.
[0914] 55. The use of any one of embodiments 52 to 54, wherein the
antibody comprised in the therapeutic agent comprises the heavy
chain variable region sequence of SEQ ID NO: 38 and the light chain
variable region sequence of SEQ ID NO: 39.
[0915] 56. The use of any one of embodiments 52 to 55, wherein the
antibody comprised in the therapeutic agent specifically binds to a
B-cell antigen, particularly an antigen selected from the group
consisting of CD20, CD19, CD22, ROR-1, CD37 and CD5, more
particularly CD20 or CD19, most particularly CD20.
[0916] 57. The use of embodiment 56, wherein the antibody comprised
in the therapeutic agent comprises a heavy chain variable region
comprising the heavy chain CDR (HCDR) 1 of SEQ ID NO: 4, the HCDR2
of SEQ ID NO: 5, and the HCDR3 of SEQ ID NO: 6; and a light chain
variable region comprising the light chain CDR (LCDR) 1 of SEQ ID
NO: 7, the LCDR2 of SEQ ID NO: 8 and the LCDR3 of SEQ ID NO: 9.
[0917] 58. The use of embodiment 56 or 57, wherein the antibody
comprised in the therapeutic agent comprises the heavy chain
variable region sequence of SEQ ID NO: 10 and the light chain
variable region sequence of SEQ ID NO: 11.
[0918] 59. The use of any one of embodiments 43 to 58, wherein the
antibody comprised in the therapeutic agent is a bispecific
antibody comprising (i) an antibody as defined in any one of
embodiments 53 to 55 and (ii) an antibody as defined in any one of
embodiments 56 to 58.
[0919] 60. The use of any one of embodiments 43 to 59, wherein the
therapeutic agent comprises CD20XCD3 bsAB.
[0920] 61. The use of any one of embodiments 43 to 51, wherein the
therapeutic agent comprises a T cell expressing a chimeric antigen
receptor (CAR), particularly a CAR that specifically binds to a
B-cell antigen, more particularly a CAR that specifically binds to
an antigen selected from the group of CD20, CD19, CD22, ROR-1, CD37
and CD5.
[0921] 62. The use of any one of embodiments 43 to 61, wherein the
disease is a B cell proliferative disorder, particularly a
CD20-positive B-cell disorder, and/or is a disease selected from
the group consisting of Non-Hodgkin lymphoma (NHL), acute
lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL),
diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL),
mantle-cell lymphoma (MCL), marginal zone lymphoma (MZL), Multiple
myeloma (MM) and Hodgkin lymphoma (HL).
[0922] 63. A kit for the reduction of cytokine release associated
with the administration of a T-cell activating therapeutic agent in
a subject, comprising a package comprising a Type II anti-CD20
antibody composition and instructions for using the Type II
anti-CD20 antibody composition in a treatment regimen
comprising
[0923] (i) administration to the subject of the Type II anti-CD20
antibody composition,
[0924] and consecutively after a period of time
[0925] (ii) administration to the subject of a T-cell activating
therapeutic agent,
[0926] wherein the period of time between the administration of the
Type II anti-CD20 antibody composition and the administration of
the therapeutic agent is sufficient for reduction of the number of
B-cells in the subject in response to the administration of the
CD20 antibody.
[0927] 64. The kit of embodiment 63, further comprising a T-cell
activating therapeutic agent composition.
[0928] 65. A kit for the treatment of a disease in a subject,
comprising a package comprising a T-cell activating therapeutic
agent composition and instructions for using the therapeutic agent
composition in a treatment regimen comprising
[0929] (i) administration to the subject of a Type II anti-CD20
antibody,
[0930] and consecutively after a period of time
[0931] (ii) administration to the subject of the T-cell activating
therapeutic agent composition,
[0932] wherein the period of time between the administration of the
Type II anti-CD20 antibody and the administration of the
therapeutic agent composition is sufficient for reduction of the
number of B-cells in the subject in response to the administration
of the CD20 antibody.
[0933] 66. The kit of embodiment 65, further comprising a Type II
anti-CD20 antibody composition.
[0934] 67. The kit of any one of embodiments 63 to 66, wherein the
treatment regimen effectively reduces cytokine release associated
with the administration of the therapeutic agent in the subject as
compared to a corresponding treatment regimen without the
administration of the Type II anti-CD20 antibody composition.
[0935] 68. The kit of any one of embodiments 63 to 67, wherein the
Type II anti-CD20 antibody comprises a heavy chain variable region
comprising the heavy chain CDR (HCDR) 1 of SEQ ID NO: 4, the HCDR2
of SEQ ID NO: 5, and the HCDR3 of SEQ ID NO: 6; and a light chain
variable region comprising the light chain CDR (LCDR) 1 of SEQ ID
NO: 7, the LCDR2 of SEQ ID NO: 8 and the LCDR3 of SEQ ID NO: 9.
[0936] 69. The kit of any one of embodiments 63 to 68, wherein the
Type II anti-CD20 antibody comprises the heavy chain variable
region sequence of SEQ ID NO: 10 and the light chain variable
region sequence of SEQ ID NO: 11.
[0937] 70. The kit of any one of embodiments 63 to 69, wherein the
Type II anti-CD20 antibody is an IgG antibody, particularly an
IgG.sub.1 antibody.
[0938] 71. The kit of any one of embodiments 63 to 70, wherein the
Type II anti-CD20 antibody is engineered to have an increased
proportion of non-fucosylated oligosaccharides in the Fc region as
compared to a non-engineered antibody.
[0939] 72. The kit of any one of embodiments 63 to 71, wherein at
least about 40% of the N-linked oligosaccharides in the Fc region
of the Type II anti-CD20 antibody are non-fucosylated.
[0940] 73. The kit of any one of embodiments 63 to 72, wherein the
Type II anti-CD20 antibody is obinutuzumab.
[0941] 74. The kit of any one of embodiments 63 to 73, wherein the
therapeutic agent comprises an antibody, particularly a
multispecific antibody.
[0942] 75. The kit of embodiment 74, wherein the antibody comprised
in the therapeutic agent specifically binds to an activating T cell
antigen, particularly an antigen selected from the group consisting
of CD3, CD28, CD137 (also known as 4-1BB), CD40, CD226, OX40, GITR,
CD27, HVEM, and CD127, more particularly CD3, most particularly
CD3.epsilon..
[0943] 76. The kit of embodiment 74 or 75, wherein the antibody
comprised in the therapeutic agent comprises a heavy chain variable
region comprising the heavy chain CDR (HCDR) 1 of SEQ ID NO: 32,
the HCDR2 of SEQ ID NO: 33, and the HCDR3 of SEQ ID NO: 34; and a
light chain variable region comprising the light chain CDR (LCDR) 1
of SEQ ID NO: 35, the LCDR2 of SEQ ID NO: 36 and the LCDR3 of SEQ
ID NO: 37.
[0944] 77. The kit of any one of embodiments 74 to 76, wherein the
antibody comprised in the therapeutic agent comprises the heavy
chain variable region sequence of SEQ ID NO: 38 and the light chain
variable region sequence of SEQ ID NO: 39.
[0945] 78. The kit of any one of embodiments 74 to 77, wherein the
antibody comprised in the therapeutic agent specifically binds to a
B-cell antigen, particularly an antigen selected from the group
consisting of CD20, CD19, CD22, ROR-1, CD37 and CD5, more
particularly CD20 or CD19, most particularly CD20.
[0946] 79. The kit of embodiment 78, wherein the antibody comprised
in the therapeutic agent comprises a heavy chain variable region
comprising the heavy chain CDR (HCDR) 1 of SEQ ID NO: 4, the HCDR2
of SEQ ID NO: 5, and the HCDR3 of SEQ ID NO: 6; and a light chain
variable region comprising the light chain CDR (LCDR) 1 of SEQ ID
NO: 7, the LCDR2 of SEQ ID NO: 8 and the LCDR3 of SEQ ID NO: 9.
[0947] 80. The kit of embodiment 78 or 79, wherein the antibody
comprised in the therapeutic agent comprises the heavy chain
variable region sequence of SEQ ID NO: 10 and the light chain
variable region sequence of SEQ ID NO: 11.
[0948] 81. The kit of any one of embodiments 78 to 80, wherein the
antibody comprised in the therapeutic agent is a bispecific
antibody comprising (i) an antibody as defined in any one of
embodiments 75 to 77 and (ii) an antibody as defined in any one of
embodiments 78 to 80.
[0949] 82. The kit of any one of embodiments 63 to 81, wherein the
therapeutic agent comprises CD20XCD3 bsAB.
[0950] 83. The kit of any one of embodiments 63 to 73, wherein the
therapeutic agent comprises a T cell expressing a chimeric antigen
receptor (CAR), particularly a CAR that specifically binds to a
B-cell antigen, more particularly a CAR that specifically binds to
an antigen selected from the group of CD20, CD19, CD22, ROR-1, CD37
and CD5.
[0951] 84. The kit of any one of embodiments 63 to 83, wherein the
disease is a B cell proliferative disorder, particularly a
CD20-positive B-cell disorder, and/or is a disease selected from
the group consisting of Non-Hodgkin lymphoma (NHL), acute
lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL),
diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL),
mantle-cell lymphoma (MCL), marginal zone lymphoma (MZL), Multiple
myeloma (MM) and Hodgkin lymphoma (HL).
[0952] 85. A T-cell activating therapeutic agent for use in a
method of treating a disease in a subject, the method comprising a
treatment regimen comprising
[0953] (i) administration to the subject of a Type II anti-CD20
antibody,
[0954] and consecutively after a period of time
[0955] (ii) administration to the subject of the T-cell activating
therapeutic agent,
[0956] wherein the period of time between the administration of the
Type II anti-CD20 antibody and the administration of the
therapeutic agent is sufficient for reduction of the number of
B-cells in the subject in response to the administration of the
CD20 antibody.
[0957] 86. The T-cell activating therapeutic agent of embodiment
85, wherein the treatment regimen effectively reduces cytokine
release in the subject associated with the administration of the
therapeutic agent as compared to a corresponding treatment regimen
without the administration of the Type II anti-CD20 antibody.
[0958] 87. The T-cell activating therapeutic agent of embodiment 85
or 86, wherein the Type II anti-CD20 antibody comprises a heavy
chain variable region comprising the heavy chain CDR (HCDR) 1 of
SEQ ID NO: 4, the HCDR2 of SEQ ID NO: 5, and the HCDR3 of SEQ ID
NO: 6; and a light chain variable region comprising the light chain
CDR (LCDR) 1 of SEQ ID NO: 7, the LCDR2 of SEQ ID NO: 8 and the
LCDR3 of SEQ ID NO: 9.
[0959] 88. The T-cell activating therapeutic agent of any one of
embodiments 85 to 87, wherein the Type II anti-CD20 antibody
comprises the heavy chain variable region sequence of SEQ ID NO: 10
and the light chain variable region sequence of SEQ ID NO: 11.
[0960] 89. The T-cell activating therapeutic agent of any one of
embodiments 85 to 88, wherein the Type II anti-CD20 antibody is an
IgG antibody, particularly an IgG.sub.1 antibody.
[0961] 90. The T-cell activating therapeutic agent of any one of
embodiments 85 to 89, wherein the Type II anti-CD20 antibody is
engineered to have an increased proportion of non-fucosylated
oligosaccharides in the Fc region as compared to a non-engineered
antibody.
[0962] 91. The T-cell activating therapeutic agent of any one of
embodiments 85 to 90, wherein at least about 40% of the N-linked
oligosaccharides in the Fc region of the Type II anti-CD20 antibody
are non-fucosylated.
[0963] 92. The T-cell activating therapeutic agent of any one of
embodiments 85 to 91, wherein the Type II anti-CD20 antibody is
obinutuzumab.
[0964] 93. The T-cell activating therapeutic agent of any one of
embodiments 85 to 92, wherein the therapeutic agent comprises an
antibody, particularly a multispecific antibody.
[0965] 94. The T-cell activating therapeutic agent of embodiment
93, wherein the antibody comprised in the therapeutic agent
specifically binds to an activating T cell antigen, particularly an
antigen selected from the group consisting of CD3, CD28, CD137
(also known as 4-1BB), CD40, CD226, OX40, GITR, CD27, HVEM, and
CD127, more particularly CD3, most particularly CD3.epsilon..
[0966] 95. The T-cell activating therapeutic agent of embodiment 93
or 94, wherein the antibody comprised in the therapeutic agent
comprises a heavy chain variable region comprising the heavy chain
CDR (HCDR) 1 of SEQ ID NO: 32, the HCDR2 of SEQ ID NO: 33, and the
HCDR3 of SEQ ID NO: 34; and a light chain variable region
comprising the light chain CDR (LCDR) 1 of SEQ ID NO: 35, the LCDR2
of SEQ ID NO: 36 and the LCDR3 of SEQ ID NO: 37.
[0967] 96. The T-cell activating therapeutic agent of any one of
embodiments 93 to 95, wherein the antibody comprised in the
therapeutic agent comprises the heavy chain variable region
sequence of SEQ ID NO: 38 and the light chain variable region
sequence of SEQ ID NO: 39.
[0968] 97. The T-cell activating therapeutic agent of any one of
embodiments 93 to 96, wherein the antibody comprised in the
therapeutic agent specifically binds to a B-cell antigen,
particularly an antigen selected from the group consisting of CD20,
CD19, CD22, ROR-1, CD37 and CD5, more particularly CD20 or CD19,
most particularly CD20.
[0969] 98. The T-cell activating therapeutic agent of embodiment
97, wherein the antibody comprised in the therapeutic agent
comprises a heavy chain variable region comprising the heavy chain
CDR (HCDR) 1 of SEQ ID NO: 4, the HCDR2 of SEQ ID NO: 5, and the
HCDR3 of SEQ ID NO: 6; and a light chain variable region comprising
the light chain CDR (LCDR) 1 of SEQ ID NO: 7, the LCDR2 of SEQ ID
NO: 8 and the LCDR3 of SEQ ID NO: 9.
[0970] 99. The T-cell activating therapeutic agent of embodiment 97
or 98, wherein the antibody comprised in the therapeutic agent
comprises the heavy chain variable region sequence of SEQ ID NO: 10
and the light chain variable region sequence of SEQ ID NO: 11.
[0971] 100. The T-cell activating therapeutic agent of any one of
embodiments 85 to 99, wherein the antibody comprised in the
therapeutic agent is a bispecific antibody comprising (i) an
antibody as defined in any one of embodiments 94 to 96 and (ii) an
antibody as defined in any one of embodiments 97 to 99.
[0972] 101. The T-cell activating therapeutic agent of any one of
embodiments 85 to 100, wherein the therapeutic agent comprises
CD20XCD3 bsAB.
[0973] 102. The T-cell activating therapeutic agent of any one of
embodiments 85 to 92, wherein the therapeutic agent comprises a T
cell expressing a chimeric antigen receptor (CAR), particularly a
CAR that specifically binds to a B-cell antigen, more particularly
a CAR that specifically binds to an antigen selected from the group
of CD20, CD19, CD22, ROR-1, CD37 and CD5.
[0974] 103. The T-cell activating therapeutic agent of any one of
embodiments 85 to 102, wherein the disease is a B cell
proliferative disorder, particularly a CD20-positive B-cell
disorder, and/or is a disease selected from the group consisting of
Non-Hodgkin lymphoma (NHL), acute lymphocytic leukemia (ALL),
chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma
(DLBCL), follicular lymphoma (FL), mantle-cell lymphoma (MCL),
marginal zone lymphoma (MZL), Multiple myeloma (MM) and Hodgkin
lymphoma (HL).
EXAMPLES
[0975] The following are examples of methods and compositions of
the invention. It is understood that various other embodiments may
be practiced, given the general description provided above.
Example 1
Prior Treatment with Obinutuzumab but not Rituximab or Vehicle
Results in the Attenuation of Tetanus Toxoid Specific De Novo IgG
Antibody Responses in Cynomolgus Monkeys
[0976] To evaluate the functional impact that B-cell depletion with
obinutuzumab or rituximab has on the humoral immune response to
foreign antigens, cynomolgus monkeys were immune challenged after
treatment with either a novel antigen that the animals had never
experienced before (de novo response to tetanus toxoid) or with a
booster immune rechallenge with an immunogen that the animals had
already encountered prior to the CD20 antibody administration
(memory recall response to measles/rubella).
[0977] Animals were administered on day -14 and day -7 rituximab or
obinutuzumab at a dose of 30 mg/kg or vehicle by i.v. infusion.
Immunization with tetanus toxoid was performed on Day 0. Naive
animals from all groups had a baseline anti-tetanus toxoid IgG
measurement of around 0.1 IU/ml at day 0. At Day 7, vehicle treated
animals and rituximab treated animals mounted robust humoral
anti-tetanus toxoid IgG responses, with an increase to around 1.0
IU/ml, while obinutuzumab treated animals showed attenuated
responses, resulting in an equal to background signal of 0.1 IU/ml.
By Day 21, serological titers in vehicle treated and rituximab
treated animals continued to rise to peak levels of 2.5 IU/ml (FIG.
1). The obinutuzumab treated animals began to display a slight
increase in titers to 0.5 IU/ml, which was significantly below of
that of vehicle and rituximab treated groups. The serum IgG
response waned by day 51 and 68 in all groups reaching around 1.5
IU/ml in vehicle, 1.3 IU/ml in rituximab and returning to 0.2 IU/ml
in obinutuzumab treated animals.
[0978] These results indicate that prior treatment with
obinutuzumab, but not rituximab, results in the attenuation of
tetanus toxoid specific de novo IgG antibody responses in
cynomolgus monkeys.
[0979] To investigate the memory recall responses by measles
specific IgG antibody production in response to immune re-challenge
with a measles/rubella booster vaccination animals that had
measurable baseline positive anti-measles titers were selected.
Animals were administered rituximab or obinutuzumab at a dose of 30
mg/kg or vehicle by i.v. infusion on day -14 and Day -7. Immune
re-challenge with a measles/rubella vaccination was performed on
Day 0. Measuring the fold change at optical density OD450 nm
reading over baseline Day -14 reading, resulted in measurable
increase in anti-measles titers in all three groups at Day 21, Day
51 and Day 68 with no significant difference found in the IgG
responses to measles amongst the three different treatment groups
(FIG. 2). The conclusion is that memory recall responses were left
intact regardless of anti-CD20 depletion therapy. The
administration of either obinutuzumab or rituximab had no
measurable impact on the memory recall responses to a booster
immunization against measles/rubella vaccination with established
basal titers as a result of prior vaccination.
[0980] Overall, these results showed that prior treatment with
obinutuzumab led to strong suppression of de novo antibody
responses, but left the protective humoral memory responses intact.
Without wishing to be bound by theory, the ability to block de novo
humoral antibody responses may possibly be attributed to either the
increased extent of endogenous B-cell depletion seen with
obinutuzumab and/or the enhanced ability of obinutuzumab to deplete
activated, CD20 expressing B cells.
Example 2
A Multi-Center, Randomized, Open-Label Phase 1 Study to Evaluate
Feasibility, Safety and Pharmacodynamic Effect of Pretreatment with
Obinutuzumab Prior to Therapy with RO6895882 (Cergutuzumab
Amunaleukin, (CEA-IL2v), in Patients with Locally Advanced and/or
Metastatic Solid Tumors
[0981] Methods
[0982] An open-label, multi-center, randomized Phase 1b clinical
sub-study of RO6895882 given with or without obinutuzumab as
pre-treatment is performed.
[0983] The main objective of this sub-study is to determine if
pre-treatment with obinutuzumab prevents the formation of ADAs in
patients treated subsequently with RO6895882.
[0984] The trial enrolls patients with locally advanced and/or
metastatic CEA-positive solid tumors that have progressed on or are
intolerant to the standard of care therapy. Obinutuzumab and
RO6895882 are administered intravenously (IV).
[0985] Fourteen patients were randomized into the obinutuzumab
pretreatment arm and five patients to the control arm without
obinutuzumab pretreatment. The patients in the obinutuzumab
pretreatment arm received 2 g of obinutuzumab, administered on two
consecutive days (2.times.1000 mg), Day -13 and Day -12 (+/-2 days)
before the Cycle 1 Day 1 (C1D1) RO6895882 administration.
Pre-medication was given prior to each obinutuzumab dosing. On C1D1
all patients received a fixed and flat 10 mg dose of RO6895882. At
subsequent cycles, all patients received 20 mg of RO6895882,
administered over a minimum of 2 hour IV infusion bi-weekly (q2W).
The patients in the control arm (without obinutuzumab pretreatment)
received RO6895882 administrations starting at C1D1 identically to
the patients in the obinutuzumab pretreatment arm.
[0986] Blood samples were collected before and during the treatment
period for the monitoring of B lymphocyte counts. B cell counts
were obtained using flow cytometry and staining for CD19. Blood
samples for ADA determination were obtained at baseline and
thereafter every second week after the RO6895882
administration.
[0987] All patients underwent baseline and on-treatment tumor
biopsies. For the obinutuzumab pretreated patients, baseline
biopsies were taken after randomization, before administration of
obinutuzumab. The control patients have tumor biopsies taken prior
to the Cycle 1 Day 1 RO6895882 administration. On-treatment tumor
biopsies from the first five patients pretreated with obinutuzumab
were collected on Cycle 1 Day 1 (+0/-1 days) prior to RO6895882
administration in order to confirm tissue B cell depletion before
the start of RO6895882 treatment. From the remaining obinutuzumab
pre-treated patients the repeated tumor biopsies were collected on
Cycle 3 Day 14 (+/-2 days), prior to the fourth RO6895882
administration on Cycle 4 Day 1. One portion of the biopsy tissue
was analyzed by flow cytometry and staining with CD19 for B
lymphocyte detection. The second portion was formalin fixed and
embedded in paraffin and analyzed for B lymphocytes using CD20 and
PAX5 staining.
[0988] The primary objectives for this study were to assess the
effect of pre-treatment with obinutuzumab on decreasing the
proportion of patients with ADA titer at cycle 4, and to evaluate
the safety and tolerability of administration of obinutuzumab prior
to treatment with RO6895882.
[0989] Secondary objectives for this study included
characterization of the RO6895882 ADAs (at cycle 4) and
investigation and characterization of the B-cell depletion in
tissue (tumor and skin) and peripheral blood resulting from
obinutuzumab pre-treatment.
[0990] Results
[0991] RO6895882 (cergutuzumab amunaleukin, CEA-IL2v) has been
tested in Study BP28920 EIH Study (ClinicalTrials.gov identifier:
NCT02004106). As of November 2016, preliminary results from Study
BP28920 show that in 59 of 74 patients (80%) anti-CEA-IL2v
antibodies were detected. Among the 59 ADA-positive patients, 58
(98%) showed a persistent immune response while 1 (1.6%) showed a
transient immune response. The intensity of ADA responses ranges
from low to high titers (10-196.830), 32 out of 59 persistent
ADA-positive cases (54%) show high ADA titers (>100). The first
onset of an immune response was observed after one dose of
806895882 (cergutuzumab amunaleukin, CEA-IL2v) as early as on day
5, i.e. 96 h post first dose. No patient with pre-existing ADAs was
identified and all ADAs were treatment induced. In five out of 59
patients (8%), in addition to high ADA titres (>100), loss of
exposure was observed. Neither hypersensitivity reactions or signs
and symptoms suggestive of hypersensitivity, nor evidence of
ADA-mediated adverse events were reported in the 108 patients who
received RO6895882.
[0992] The above-described clinical sub-study was initiated to
explore the potential of obinutuzumab (Gazyva.RTM.) given as a
pre-treatment to administration of RO6895882. (cergutuzumab
amunaleukin, CEA-IL2v) to attenuate development of anti-drug
antibodies (ADAs). The study has concluded.
[0993] Fourteen out of 14 obinutuzumab treated patients remained
free of ADAs against RO6895882 on Cycle 2 Day 1 (two weeks after
first RO6895882 administration) and throughout the entire study
period. The longest on-treatment follow up period was up to 10
treatment cycles, i.e. 20 weeks. Four out of five control patients
who were not pre-treated with obinutuzumab developed ADAs with
titers of 10 to 270 after 1 dose of RO6895882.
[0994] The safety profile of obinutuzumab pre-treatment was
acceptable in patients with locally advanced and/or metastatic
solid tumors. Patients receiving obinutuzumab/CEA-IL2v treatment
did not experience unexpected AEs compared to the CEA-IL2v
monotherapy. No fatal outcome due to adverse event was
reported.
[0995] Flow cytometric analysis of peripheral blood indicated
complete depletion of B cells after obinutuzumab treatment. Before
the start of treatment with RO6895882 (C1D1), no B cells were
detectable in the collected blood samples (FIG. 3).
[0996] The tumor biopsy samples from obinutuzumab pre-treatment
patients were analyzed by both flow cytometry and
immunohistochemistry (IHC) for the frequency of B cells. Flow
cytometric analysis (FIG. 4) showed a strong reduction in the
number and percentage of cells that stain positive for CD19, a
surface protein expressed on B lymphocytes. IHC analysis of a
different portion of the paired tumor biopsy samples detected a
decrease in cells staining positive for CD20, a second antigen
expressed on B cells (FIG. 5 A and B). In order to exclude that the
reduction in CD20 staining was caused by obinutuzumab which may
compete with anti-CD20 staining, the results were confirmed by
staining with PAX5, a B cell specific transcription factor, which
was also strongly reduced (FIG. 5 C and D). Taken together, these
results indicate an effective reduction of B cells in the tumor and
adjacent normal tissue.
[0997] In conclusion, the above data from this sub-study strongly
suggest that obinutuzumab is efficacious in mitigating ADAs.
Example 3
Clinical Evaluation of Feasibility, Safety and Pharmacodynamic
Effect of Pre-Treatment with Obinutuzumab Prior to Therapy with CEA
TCB
[0998] An ongoing phase I clinical trial with CEA TCB
(ClinicalTrials.gov identifier: NCT02324257) enrolls patients with
locally advanced and/or metastatic CEA-positive solid tumors who
have progressed on standard treatment, are intolerant to standard
of care (SOC), and/or are non-amenable to SOC.
[0999] Parallel cohorts of patients are opened to enroll patients
that will be pretreated with obinutuzumab. The main objective of
these cohorts is to determine if pre-treatment with obinutuzumab
prevents the formation of ADAs in patients treated subsequently
with CEA TCB. Obinutuzumab and CEA TCB are administered
intravenously (IV). The patients in the obinutuzumab pre-treatment
cohorts receive 2000 mg of obinutuzumab, either 2000 mg of
obinutuzumab IV on Day-13 or 1000 mg of obinutuzumab IV on two
consecutive days, Day-13 and Day-12 (.+-.2 days) before the Cycle 1
Day 1 (C1D1) of CEA TCB administration. Pre-medication including
analgesic, anti-histamine and corticosteroid is given prior to each
obinutuzumab dosing. On C1D1, all patients receive a flat dose
(depending on the dose cohort they are allocated to, as defined in
the ongoing clinical trial) of CEA TCB over a minimum of 120-minute
IV infusion. At subsequent cycles, all patients receive the same
dose of CEA TCB, administered over a minimum of 90-minute IV
infusion at C2D1 and over a minimum of 60-minute IV infusion from
C3D1 onwards weekly (QW). The patients not receiving obinutuzumab
pre-treatment receive CEA TCB administrations starting at C1D1
identically to the patients in the obinutuzumab pre-treatment
cohorts. Blood samples are collected before and during the
treatment period for the monitoring of B lymphocyte counts. B cell
counts are obtained using flow cytometry and staining for CD19.
Blood samples for PK to evaluate the serum levels of CEA TCB and
for ADA determination are obtained at baseline and thereafter at
each cycle of CEA TCB administration. The primary objectives for
these cohorts of patients receiving obinutuzumab pre-treatment is
the effect of obinutuzumab pretreatment in decreasing the rate of
patients with positive Anti-Drug Antibodies (ADA) titer against CEA
TCB at week 8 and/or delaying the time of onset of ADA against CEA
TCB, and to evaluate the safety and tolerability of administration
of obinutuzumab prior to treatment with CEA TCB. The study will
also be looking at the characterization of the ADA directed against
CEA TCB; it includes as well investigation and characterization of
the B-cell depletion in tissue (tumor biopsies are undertaken at
Baseline and at week 7 after C1D1 with CEA TCB) and peripheral
blood resulting from obinutuzumab pre-treatment.
[1000] Preliminary Results
[1001] CEA TCB is being tested in a EIH Study (ClinicalTrials.gov
identifier: NCT02324257). As of 27 Oct. 2016 (ADA data cutoff),
preliminary results from Study BP29541 show that in 40 of 77
patients (52%) anti-CEA TCB antibodies were detected, none of these
patients received obinutuzumab pre-treatment, except for two
patients with transient positive titers (one had a titer of 30 at
Cycle 2 Day 1 but became then negative up to Cycle 24 and another
one had a titer of 270 at Cycle 3 Day 1 and 90 at Cycle 4 Day 1 but
became then negative up to Cycle 8). Except for these two patients,
all the 38 other ADA-positive patients showed a persistent immune
response. The intensity of ADA responses ranged from low to high
titers (10-21870). 17 out of 38 persistent ADA-positive cases (45%)
showed moderate ADA titers (.ltoreq.810), while 21 out of 28
persistent ADA-positive cases (55%) showed high ADA titers
(>810). The first onset of an immune response was observed after
one dose of CEA TCB at day 8 (C2D1 pre-dose), however 3 patients
had positive titers at Cycle 1 Day 1 pre-dose. Three patients with
pre-existing ADAs were identified and in all other patients ADAs
were treatment-induced. In 24 out of 38 patients (63%) with a
persistent immune response, the sustained ADA presence correlated
with total loss of exposure (ranges from Cycle 3 Day 1 and Cycle 13
Day 1 (PK data cutoff: 7 Nov. 2016)). So far no hypersensitivity
reactions or signs and symptoms suggestive of hypersensitivity, or
evidence of ADA-mediated adverse events have been reported in the
77 patients who received CEA TCB. The above-described clinical
study with parallel cohorts of patients receiving obinutuzumab
pre-treatment was initiated to explore the potential of
obinutuzumab (Gazyva.RTM./Gazyvaro.RTM.) given as a pre-treatment
to administration of CEA TCB to attenuate development of anti-drug
antibodies (ADAs). The study is ongoing. According to the
preliminary data, as of the clinical cutoff date of 27 Oct. 2016
(ADA data cutoff), the 12 obinutuzumab pre-treated patients for
which ADA data is available so far (2 of the patients only received
obinutuzumab as they clinically deteriorated in the meantime and
did not receive any CEA TCB infusion) remained free of ADAs against
CEA TCB at the following time points: 1 patient up to C1, 1 patient
up to C3, 1 patient up to C4, 1 patient up to C6, 1 patient up to
C8, 1 patient up to C10, 2 patients up to C12, 1 patient up to C16,
and 1 patient up to C25.
[1002] Eight out of sixty-five patients who were not pre-treated
with obinutuzumab developed ADAs with titers of 10 to 810 after 1
dose of CEA TCB. Based on a preliminary safety analysis, the safety
profile of obinutuzumab pre-treatment was acceptable in patients
with locally advanced and/or metastatic solid tumors. Patients
receiving obinutuzumab pre-treatment prior to receiving weekly
doses of CEA TCB did not experience unexpected adverse events (AEs)
compared to the ones who did not receive obinutuzumab
pre-treatment. Flow cytometric analysis of peripheral blood
indicated complete depletion of B cells after obinutuzumab
treatment. Before the start of treatment with CEA TCB (C1D1), no B
cells were detectable in the collected blood samples. The tumor
biopsy samples from obinutuzumab pre-treatment patients were
undertaken at Baseline prior to receiving obinutuzumab
pre-treatment and on treatment at Cycle 7 Day 1. The analyses are
still ongoing.
Example 4
Assessment of the Anti-Tumour Activity and Cytokine Release
Mediated by CD20XCD3 bsAB.+-.Obinutuzumab Pre-Treatment (Gpt) in
Fully Humanized Mice
[1003] We investigated whether Gpt could prevent the cytokine
release associated with the first administration of CD20XCD3 bsAB
in fully humanized NOG mice.
[1004] All treatment options (obinutuzumab, CD20XCD3 bsAB and
Gpt+CD20XCD3 bsAB) led to efficient peripheral blood B-cell
depletion detected already 24 hours after the first therapy
administration (FIG. 7A). T cell counts revealed a transient
decrease in the peripheral blood 24 hours after the first
administration of CD20XCD3 bsAB but not following obinutuzumab or
Gpt+CD20XCD3 bsAB (FIG. 7B). Therefore, when administered prior to
CD20XCD3 bsAB, a single administration of obinutuzumab abrogates
CD20XCD3 bsAB-mediated T cell decrease in the peripheral blood.
[1005] The analysis of cytokines released in blood of treated mice
in the different experimental groups revealed that CD20XCD3 bsAB
treatment induces a transient elevation of several cytokines in the
blood, with a peak at 24 hours after the first administration and a
return to near baseline levels by 72 hours (FIG. 8). MIP-1b, IL-5,
IL-10, MCP-1 show a similar trend to IFN.gamma., TNF.alpha. and
IL-6 (not shown). Gpt strongly reduced the cytokine release in the
peripheral blood associated with the first CD20XCD3 bsAB injection
(Table 2).
TABLE-US-00002 TABLE 2 Cytokines Released in the Peripheral Blood
of Fully Humanized NOG Mice upon CD20XCD3 bsAB and Gpt + CD20XCD3
bsAB Treatments Treatment CD20XCD3 Gpt + CD20XCD3 Vehicle bsAB bsAB
Cytokine (pg/ml) (pg/ml) (pg/ml) IFN-g 18.50 (18.07) 756.95
(357.30) 183.134 (171.91) TNF-a 12.47 (2.95) 79.56 (28.98) 14.89
(2.56) IL-6 15.39 (7.15) 613.27 (140.60) 178.34 (117.85) IL-8 11.44
(2.64) 292.68 (132.36) 150.58 (96.76) MIP-1b 272.70 (97.05) 2129.44
(132.36) 338.95 (71.25) MCP-1 73.49 (13.89) 2146.31 (672.69) 393.29
(188.86) IL-10 223.48 (62.48) 15,278.89 (6584.50) 945.04 (604.89)
IL-4 0.75 (0.14) 1.99 (0.77) 0.81 (0.02) G-CSF 14.60 (5.14) 21.23
(16.36) 3.82 (2.02) GM-CSF 945.97 (155.74) 1207.48 (299.83) 626.18
(282.46) IL-5 10.42 (3.35) 162.33 (140.82) 13.58 (8.44) IL-2 19.1
(8.42) 369.70 (360.64) 19.59 (17.64) IL-13 5.39 (3.66) 15.42
(11.18) 2.96 (1.11) IL-1b 1.48 (0.2) 6.40 (1.94) 3.47 (1.88) IL-7
6.98 (0) 4.27 (2.55) 6.17 (1.79) IL-12p40 43.59 (19.45) 51.31
(23.12) 17.05 (2.62) IL-17 194.40 (96.32) 274.79 (112.20) 73.33
(32.43) Notes: Data are displayed as the arithmetic mean (SD). N =
5 in both treatments.
[1006] The anti-tumour activity of CD20XCD3 bsAB was not affected
by pre-treatment with obinutuzumab (FIG. 9). Obinutuzumab
treatment, as monotherapy, showed a strong anti-tumour activity,
although with slower kinetics when compared to CD20XCD3 bsAB in
this tumour and mouse model.
[1007] The data therefore indicate that Gpt reduces the cytokine
release associated with the first CD20XCD3 bsAB injection, however,
despite targeting the same antigen on tumour cells, the anti-tumour
activity of CD20XCD3 bsAB is not affected by Gpt.
Example 5
Obinutuzumab Pre-Treatment Study in Cynomolgus Monkeys
[1008] A mechanistic study (non-GLP) in male cynomolgus monkeys was
performed to investigate the effects of pretreatment with
obinutuzumab on response to CD20XCD3 bsAB at doses of 0.1, 0.3 and
1 mg/kg) (Table 3). In this study, 6 naive cynomolgus male
monkeys/group (4 for Group 1), received an IV dose of either
control article 1 (Groups 1 and 2) or obinutuzumab (50 mg/kg,
Groups 3, 4, 5), followed 4 days later by treatment with control
article 2 (Group 1), CD20XCD3 bsAB, 0.1 mg/kg (Group 2, Group 3),
CD20XCD3 bsAB, 0.3 mg/kg (Group 4) or CD20XCD3 bsAB, 1 mg/kg (Group
5). Four days between the obinutuzumab and CD20XCD3 bsAB dosing was
considered sufficient to allow depletion of B cells in peripheral
blood, lymph nodes and spleen by obinutuzumab. On Day 12, 2 animals
from Group 1, and 4 from Groups 2 to 5 were necropsied (terminal
necropsy). Two animals from each group were retained for an 8-week
recovery period.
TABLE-US-00003 TABLE 3 Study Design: Obinutuzumab Pre- Treatment in
Cynomolgus Monkeys. Dose Group Dosing Level Number of Males No.
Test Article Day (mg/kg) Main .sup.a Recovery .sup.b 1 Control
Article 1 1 0 2 2 Control Article 2 5 0 2 Control Article 1 1 0 4 2
CD20XCD3 bsAB 5 0.1 3 obinutuzumab 1 50 4 2 CD20XCD3 bsAB 5 0.1 4
obinutuzumab 1 50 4 2 CD20XCD3 bsAB 5 0.3 5 obinutuzumab 1 50 4 2
CD20XCD3 bsAB 5 1 Note: Control Article 1 = Control for
obinutuzumab: Control article 2 = Control for CD20XCD3 bsAB. .sup.a
Main group animals, terminal necropsy Day 12. .sup.b Recovery
animals, necropsy week 8 (Day 61).
[1009] The following data are available from this study: [1010]
Following pretreatment with obinutuzumab (50 mg/kg, Gpt), IV
administration of CD20XCD3 bsAB was tolerated up to 1 mg/kg, the
highest tested dose. Clinical signs, observed with CD20XCD3 bsAB
alone (emesis, hunched posture and hypoactivity) were markedly
reduced by Gpt at all doses of CD20XCD3 bsAB. [1011] CD20XCD3 bsAB
administration alone resulted in the reduction of B lymphocytes and
the activation and expansion of T-lymphocyte (CD4+ and CD8+)
subsets and NK cells. In contrast, the administration of
obinutuzumab prior to CD20XCD3 bsAB administration resulted in
B-lymphocyte depletion, as well as the subsequent attenuation of
T-lymphocyte activation as demonstrated by reductions in the
transient reductions of lymphocyte and monocyte populations after
CD20XCD3 bsAB administration, as well as reductions in T-cell
activation marker up-regulation and expansion, relative to changes
present for animals that were treated with CD20XCD3 bsAB alone.
[1012] The release of IFN.gamma., IL-8, TNF.alpha., IL-2 and IL-6,
4-hour post-0.1 mg/kg CD20XCD3 bsAB treatment, was markedly reduced
in the Gpt groups. Similarly, low levels of cytokine release were
noted at higher doses of CD20XCD3 bsAB in Gpt groups (FIG. 10).
[1013] CD20XCD3 bsAB-related histopathologic findings were
restricted to the lymphoid organs (e.g. decreased cellularity
specifically affecting the CD20-positive cells was present in the
lymphoid follicles of the spleen). The CD20-positive cell decreases
were almost completely reversed after the 8 week treatment-free
period. No other histopathological changes were present, including
in brain, spinal cord and sciatic nerve in monkeys treated with
CD20XCD3 bsAB at 0.1 mg/kg and in animals administered CD20XCD3
bsAB at 0.1, 0.3 or 1 mg/kg following Gpt.
Example 6
Clinical Evaluation of Safety, Tolerability and Pharmacokinetics of
CD20XCD3 bsAB with Obinutuzumab Pre-Treatment in Patients with r/r
NHL
[1014] A phase I dose-escalation study will be performed, the
primary objectives of which include evaluation of the safety,
tolerability and pharmacokinetics CD20XCD3 bsAB with obinutuzumab
pre-treatment in patients with relapsed/refractory (r/r) NHL.
[1015] The study will enroll patients with r/r NHL, whose tumours
are expected to express CD20 in B cells. Patients with CLL will not
be enrolled. Patients are expected to have relapsed after or failed
to respond to at least one prior treatment regimen.
[1016] Obinutuzumab and CD20XCD3 bsAB will be administered
intravenously (IV).
[1017] Prior to administration of obinutuzumab and CD20XCD3 bsAB,
premedication with corticosteroids (e.g., 20 mg IV dexamethasone,
80 mg IV methylprednisone, or equivalent) will be administered,
along with anti-histamines and acetaminophen. Prophylactic measures
for other events, such as tumor lysis syndrome will also be either
recommended as needed or mandated.
[1018] CD20XCD3 bsAB will be initiated on Cycle 1/Day 1 (C1/D1) as
a single agent by intravenous (IV) infusion, following
pre-treatment with a single dose of obinutuzumab (1000 mg; IV)
seven days in advance (Cycle 1/Day -7) of the first CD20XCD3 bsAB
dose (Cycle 1/Day 1). The anticipated starting dose of CD20XCD3
bsAB will be 5 micrograms (flat dosing). All dosing cycles are 14
days (Q2W) long, with one additional dose given in Cycle 1 only.
Thus, the dosing scheme is for administration of CD20XCD3 bsAB on
Days 1 and 8 in Cycle 1 (C1/D1; C1/D8), followed by dosing in all
subsequent Cycles on Day 1 only (Q2W) for a total of 12 cycles (24
weeks) of treatment or until unacceptable toxicity or progression
occurs.
[1019] Blood samples will be collected at appropriate timepoints to
determine the relevant PK properties of CD20XCD3 bsAB, as well as a
range of PD markers in blood, to assess e.g. magnitude and kinetics
of B-cell depletion following Gpt and CD20XCD3 bsAB dose
initiation, T-cell phenotypes, and to assess soluble mediator
release (cytokines and chemokines), following administration of Gpt
and CD20XCD3 bsAB at selected timepoints.
Example 7
Comparison of the Anti-Tumor Activity of Step-Up Dosing (SUD) and
Obinutuzumab Pretreatment (Gpt)
[1020] As shown in this example, Gpt is a superior approach to
step-up dosing (SUD) in terms of anti-tumor activity and T-cell
redistribution in peripheral tissues.
[1021] Fully humanized NOG female mice (Taconic) were generated in
house. Age was 20 weeks at start of experiment. All mice were
injected s.c. on study day 0 with 1.5.times.10.sup.6 of WSU-DLCL2
cells (human diffuse large B cell lymphoma). Seven days after tumor
cell administration (Day 7), mice were administered i.v. with the
first treatment as indicated in Table 4. The second treatment as
indicated in Table 4 was administered on Day 14.
[1022] Following 9 weeks of CD20XCD3 bsAB treatment, the number of
tumor-free mice at study termination (Day 69) was higher when
treatment was preceded by obinutuzumab (Gpt) than when preceded by
any of three single fractionated doses of CD20XCD3 bsAB (SUD) (FIG.
11, Table 4).
TABLE-US-00004 TABLE 4 Anti-tumor activity upon step-up dosing of
CD20XCD3 BSAB and obinutuzumab pretreatment (Gpt) in fully
humanized NOG mice bearing WSU-DLCL2 tumors. Tumor-Free Mice at
1.sup.st Treatment (Dose) 2.sup.nd Treatment (Dose) Termination
(Day 69) CD20XCD3 bsAB CD20XCD3 bsAB 4/9 (0.15 mg/kg) (0.5 mg/kg)
CD20XCD3 bsAB CD20XCD3 bsAB 2/9 (0.05 mg/kg) (0.5 mg/kg) CD20XCD3
bsAB CD20XCD3 bsAB 3/10 (0.015 mg/kg) (0.5 mg/kg) Obinutuzumab
CD20XCD3 bsAB 7/10 (10 mg/kg) (0.5 mg/kg)
[1023] In addition to superior anti-tumor efficacy, seven days
following the first (pre-)treatment (corresponding to study Day
14), an increase in perivascular CD3 positive T cells was observed
in the lungs of CD20XCD3 bsAB-treated mice (full dose or fraction
of the full dose) but not with obinutuzumab (10 mg/kg)-treated mice
(FIG. 12 A-D). The same was true for the analysis at a later time
point corresponding to 24 hours after the second treatment (study
Day 15) (FIG. 12 E-H).
[1024] For this experiment, mice were generated and injected with
tumor cells as described above. Seven days after tumor cell
administration, mice were administered i.v. with the treatments as
indicated in Table 5A.
[1025] Four animals/group were sacrificed 7 days after receiving a
single dose of CD20XCD3 bsAB, obinutuzumab or vehicle. The
remaining four animals/group were sacrificed 24 hours after
receiving the second treatment on Day 14, as indicated in Table 5B.
Controls received the vehicle buffer.
[1026] Lung, liver and kidney were collected at necropsy and serial
sections were stained with immunohistochemistry (IHC) for human CD3
according to established protocols. Sections stained
immunohistochemically for CD3 were evaluated blinded by a Board
Certified Veterinary Pathologist using a score from 0 (no or rare
CD3 positive cells in the section) to 3 (many CD3 positive cells
surrounding the vessels/ducts).
[1027] Representative results from lung sections are shown in FIG.
12, Table 5.
[1028] A dose dependent increase in perivascular CD3 positive T
cells was observed in the lung of mice seven days after single dose
of .gtoreq.0.05 mg/kg CD20XCD3 bsAB (full dose or fraction of the
full dose), but not in obinutuzumab-treated mice (FIG. 12 A-D,
Table 5A).
TABLE-US-00005 TABLE 5A Lung IHC for CD3 - animals sacrificed 7
days after first treatment. Table shows the average score of
perivascular CD3 positive T cells in the lung (performed by blinded
pathologist analysis). Treatment (dose) Average Score Vehicle (-)
0.75 CD20XCD3 bsAB 3 (0.5 mg/kg) CD20XCD3 bsAB 1.75 (0.15 mg/kg)
CD20XCD3 bsAB 1.5 (0.05 mg/kg) CD20XCD3 bsAB 0.5 (0.015 mg/kg)
Obinutuzumab 0.25 (10 mg/kg)
[1029] An increase in perivascular T cells was also observed in
mice 24 hours after a single or a repeated dose of CD20XCD3 bsAB,
but not in mice pretreated with obinutuzumab (FIG. 12 E-H, Table
5B).
TABLE-US-00006 TABLE 5B Lung IHC for CD3 - animals sacrificed 24
hours after 2nd treatment. Table shows the average score of
perivascular CD3 positive T cells in the lung (performed by blinded
pathologist analysis). First Treatment Second Treatment (dose)
(dose) Average Score Vehicle (-) Vehicle (-) 0 Vehicle (-) CD20XCD3
bsAB 1.5 (0.5 mg/kg) CD20XCD3 bsAB CD20XCD3 bsAB 1.7 (0.15 mg/kg)
(0.5 mg/kg) CD20XCD3 bsAB CD20XCD3 bsAB 2.5 (0.05 mg/kg) (0.5
mg/kg) CD20XCD3 bsAB CD20XCD3 bsAB 2.3 (0.015 mg/kg) (0.5 mg/kg)
Obinutuzumab CD20XCD3 bsAB 0.5 (10 mg/kg) (0.5 mg/kg)
[1030] In animals receiving a single dose of 0.5 mg/kg of CD20XCD3
bsAB sacrificed 24 hours after treatment, margination and adhesion
of CD3 positive T cells was present in lung vessels (FIG. 13),
suggesting an early activation of T cells with transmigration into
the perivascular space.
[1031] In conclusion, single or repeated treatment with full dose
or fraction of the full dose of CD20XCD3 bsAB resulted in an
increase in CD3 positive T cells compared to controls with
perivascular distribution in the lung, perivascular/periductal in
the liver and in the glomeruli in the kidneys (liver and kidney
data not shown). These changes were not occurring in animals
pretreated with obinutuzumab.
Example 8
Comparison of CD20XCD3 bsAB Exposure in Cynomolgus Monkeys with or
without Gpt
[1032] In this example, Gpt is shown to increase CD20XCD3 bsAB
exposure. Cynomolgus monkeys were administered a single IV dose of
100-1000 .mu.g/kg CD20XCD3 bsAB with or without obinutuzumab
pretreatment (Gpt) (50 mg/kg, 4 days prior to CD20XCD3 bsAB
administration).
[1033] The increased exposure resulting from the depletion of
CD20.sup.+ B cells by obinutuzumab (50 mg/kg) is shown by the
CD20XCD3 bsAB serum levels following a single dose of 100 .mu.g/kg
CD20XCD3 bsAB with or without Gpt (FIG. 14). Clearance of CD20XCD3
bsAB following Gpt was markedly reduced to about one fourth of the
clearance observed in the group without Gpt (Table 6). By the time
of CD20XCD3 bsAB dosing, B cells were reduced by Gpt to about 1-5%
of baseline levels. The reduction in clearance is consistent with
the Gpt-induced B-cell depletion and the resulting reduction of
target-mediated clearance from B-cell binding. Following Gpt,
clearance and the central volume of distribution (Vc) were
independent of dose in the dose range studied, with clearance
values around 90 mL/day/kg and Vc values around 40 mL/kg, which is
similar to the serum volume.
TABLE-US-00007 TABLE 6 Mean Pharmacokinetic parameters of CD20XCD3
bsAB following a single intravenous administration with or without
obinutuzumab pretreatment in cynomolgus monkeys. Dose Obinutuzumab
CL V.sub.c T.sub.1/2.sup.a C.sub.max AUC.sub.inf/Dose (.mu.g/kg)
pretreatment (mL/day/kg) (mL/kg) (h) (.mu.g/mL) (.mu.g
h/mL/.mu.g/kg) 100 No 384 43.2 7.53 2.22 0.0679 (27.7) (16.8)
(50.3) (17.3) (36.3) 100 Yes 81.7 41.1 67.7 2.56 0.308 (22.4)
(13.6) (34.0) (12.5) (25.1) 300 Yes 110 37.9 53.4 7.63 0.234 (28.8)
(5.2) (10.4) (7.8) (29.4) 1000 Yes 87.5 34.9 54.6 28.7 0.283 (19.0)
(10.0) (17.6) (9.8) (19.7) AUC.sub.inf = area under the
concentration-time curve from Time 0 to infinity; CL = clearance:
C.sub.max = maximum concentration; T.sub.1/2 = half-life; V.sub.c =
central volume of distribution. Notes: Cynomolgus monkeys were
administered a single IV dose of 100-1000 .mu.g/kg CD20XCD3 bsAB
with or without obinutuzumab pretreatment (Gpt) (50 mg/kg, 5 days
prior to CD20XCD3 bsAB administration) in a Mechanistic Safety
Study. Values are averages of six animals per group (.+-.% CV).
.sup.aApparent T.sub.1/2 (due to non-linear pharmacokinetics)
Example 9
Obinutuzumab Pre-Treatment to Avoid Cytokine Release after Adoptive
T Cell Therapy with CAR-T Cells
[1034] Cytokine release syndrome (CRS) is a very frequent
phenomenon following treatment with CD19 CAR-T cells as well as
CAR-T cells directed against CD20 or CD22 that can result in lethal
side effects. Strategies to avoid or reduce CRS focus on various
aspects of CAR-T therapy (reviewed in Xu and Tang, Cancer Letters
(2014) 343, 172-178).
[1035] We suggest a novel approach to avoid CRS following treatment
with CAR-T cells in B cell proliferative disorders, by depletion of
peripheral and malignant B cells using obinutuzumab
pre-treatment.
[1036] For this purpose, patients with a B-cell proliferative
disorder (e.g. NHL) are randomized into an obinutuzumab
pre-treatment arm and a control arm without obinutuzumab
pre-treatment. The patients in the obinutuzumab pre-treatment arm
receive 1 g of obinutuzumab, administered on Day -7 (+/-2 days)
before administration of CD19, CD20 or CD22 CAR-T cells.
[1037] Patients are infused with autologous T cells transduced with
a CAR lentiviral vector at an appropriate dose for the specific
CAR-T cell used, the patient and the disease to be treated (e.g.
0.76.times.10.sup.6 to 20.6.times.10.sup.6 CAR-T cells per kilogram
of body weight as described in Maude et al., N Engl J Med (2014)
371,1507-1517; 1.4.times.10.sup.6 to 1.2.times.10.sup.7 CAR-T cells
per kilogram of body weight as described in Grupp et al., New Engl
J Med (2013) 368, 1509-1518; or 0.14.times.10.sup.8 to
11.times.10.sup.8 CAR-T cells as described in Porter et al., Sci
Transl Med (2015) 7, 303ra139). Patients are monitored for a
response, toxic effects, and the expansion and persistence of
circulating CAR-T cells.
[1038] Pre-medication is given prior to each obinutuzumab dosing.
Blood samples are collected before and during the treatment period
for the monitoring of B lymphocyte counts. B cell counts are
obtained using flow cytometry and staining for CD19. In addition,
incidence of CRS is screened by measuring cytokines including
IL-6.
[1039] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, the descriptions and examples should not be
construed as limiting the scope of the invention. The disclosures
of all patent and scientific literature cited herein are expressly
incorporated in their entirety by reference.
Sequence CWU 1
1
1501297PRTHomo sapiens 1Met Thr Thr Pro Arg Asn Ser Val Asn Gly Thr
Phe Pro Ala Glu Pro1 5 10 15Met Lys Gly Pro Ile Ala Met Gln Ser Gly
Pro Lys Pro Leu Phe Arg 20 25 30Arg Met Ser Ser Leu Val Gly Pro Thr
Gln Ser Phe Phe Met Arg Glu 35 40 45Ser Lys Thr Leu Gly Ala Val Gln
Ile Met Asn Gly Leu Phe His Ile 50 55 60Ala Leu Gly Gly Leu Leu Met
Ile Pro Ala Gly Ile Tyr Ala Pro Ile65 70 75 80Cys Val Thr Val Trp
Tyr Pro Leu Trp Gly Gly Ile Met Tyr Ile Ile 85 90 95Ser Gly Ser Leu
Leu Ala Ala Thr Glu Lys Asn Ser Arg Lys Cys Leu 100 105 110Val Lys
Gly Lys Met Ile Met Asn Ser Leu Ser Leu Phe Ala Ala Ile 115 120
125Ser Gly Met Ile Leu Ser Ile Met Asp Ile Leu Asn Ile Lys Ile Ser
130 135 140His Phe Leu Lys Met Glu Ser Leu Asn Phe Ile Arg Ala His
Thr Pro145 150 155 160Tyr Ile Asn Ile Tyr Asn Cys Glu Pro Ala Asn
Pro Ser Glu Lys Asn 165 170 175Ser Pro Ser Thr Gln Tyr Cys Tyr Ser
Ile Gln Ser Leu Phe Leu Gly 180 185 190Ile Leu Ser Val Met Leu Ile
Phe Ala Phe Phe Gln Glu Leu Val Ile 195 200 205Ala Gly Ile Val Glu
Asn Glu Trp Lys Arg Thr Cys Ser Arg Pro Lys 210 215 220Ser Asn Ile
Val Leu Leu Ser Ala Glu Glu Lys Lys Glu Gln Thr Ile225 230 235
240Glu Ile Lys Glu Glu Val Val Gly Leu Thr Glu Thr Ser Ser Gln Pro
245 250 255Lys Asn Glu Glu Asp Ile Glu Ile Ile Pro Ile Gln Glu Glu
Glu Glu 260 265 270Glu Glu Thr Glu Thr Asn Phe Pro Glu Pro Pro Gln
Asp Gln Glu Ser 275 280 285Ser Pro Ile Glu Asn Asp Ser Ser Pro 290
2952112PRTMus musculus 2Gly Pro Glu Leu Val Lys Pro Gly Ala Ser Val
Lys Ile Ser Cys Lys1 5 10 15Ala Ser Gly Tyr Ala Phe Ser Tyr Ser Trp
Met Asn Trp Val Lys Leu 20 25 30Arg Pro Gly Gln Gly Leu Glu Trp Ile
Gly Arg Ile Phe Pro Gly Asp 35 40 45Gly Asp Thr Asp Tyr Asn Gly Lys
Phe Lys Gly Lys Ala Thr Leu Thr 50 55 60Ala Asp Lys Ser Ser Asn Thr
Ala Tyr Met Gln Leu Thr Ser Leu Thr65 70 75 80Ser Val Asp Ser Ala
Val Tyr Leu Cys Ala Arg Asn Val Phe Asp Gly 85 90 95Tyr Trp Leu Val
Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala 100 105
1103103PRTMus musculus 3Asn Pro Val Thr Leu Gly Thr Ser Ala Ser Ile
Ser Cys Arg Ser Ser1 5 10 15Lys Ser Leu Leu His Ser Asn Gly Ile Thr
Tyr Leu Tyr Trp Tyr Leu 20 25 30Gln Lys Pro Gly Gln Ser Pro Gln Leu
Leu Ile Tyr Gln Met Ser Asn 35 40 45Leu Val Ser Gly Val Pro Asp Arg
Phe Ser Ser Ser Gly Ser Gly Thr 50 55 60Asp Phe Thr Leu Arg Ile Ser
Arg Val Glu Ala Glu Asp Val Gly Val65 70 75 80Tyr Tyr Cys Ala Gln
Asn Leu Glu Leu Pro Tyr Thr Phe Gly Gly Gly 85 90 95Thr Lys Leu Glu
Ile Lys Arg 10045PRTArtificial SequenceCD20 HCDR1 4Tyr Ser Trp Ile
Asn1 5517PRTArtificial SequenceCD20 HCDR2 5Arg Ile Phe Pro Gly Asp
Gly Asp Thr Asp Tyr Asn Gly Lys Phe Lys1 5 10 15Gly610PRTArtificial
SequenceCD20 HCDR3 6Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr1 5
10716PRTArtificial SequenceCD20 LCDR1 7Arg Ser Ser Lys Ser Leu Leu
His Ser Asn Gly Ile Thr Tyr Leu Tyr1 5 10 1587PRTArtificial
SequenceCD20 LCDR2 8Gln Met Ser Asn Leu Val Ser1 599PRTArtificial
SequenceCD20 LCDR3 9Ala Gln Asn Leu Glu Leu Pro Tyr Thr1
510119PRTArtificial SequenceCD20 VH 10Gln Val Gln Leu Val Gln Ser
Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys
Lys Ala Ser Gly Tyr Ala Phe Ser Tyr Ser 20 25 30Trp Ile Asn Trp Val
Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Arg Ile Phe
Pro Gly Asp Gly Asp Thr Asp Tyr Asn Gly Lys Phe 50 55 60Lys Gly Arg
Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr65 70 75 80Met
Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95Ala Arg Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr Trp Gly Gln Gly
100 105 110Thr Leu Val Thr Val Ser Ser 11511115PRTArtificial
SequenceCD20 VL 11Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Pro
Val Thr Pro Gly1 5 10 15Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Lys
Ser Leu Leu His Ser 20 25 30Asn Gly Ile Thr Tyr Leu Tyr Trp Tyr Leu
Gln Lys Pro Gly Gln Ser 35 40 45Pro Gln Leu Leu Ile Tyr Gln Met Ser
Asn Leu Val Ser Gly Val Pro 50 55 60Asp Arg Phe Ser Gly Ser Gly Ser
Gly Thr Asp Phe Thr Leu Lys Ile65 70 75 80Ser Arg Val Glu Ala Glu
Asp Val Gly Val Tyr Tyr Cys Ala Gln Asn 85 90 95Leu Glu Leu Pro Tyr
Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110Arg Thr Val
11512133PRTHomo sapiens 12Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr
Gln Leu Gln Leu Glu His1 5 10 15Leu Leu Leu Asp Leu Gln Met Ile Leu
Asn Gly Ile Asn Asn Tyr Lys 20 25 30Asn Pro Lys Leu Thr Arg Met Leu
Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45Lys Ala Thr Glu Leu Lys His
Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60Pro Leu Glu Glu Val Leu
Asn Leu Ala Gln Ser Lys Asn Phe His Leu65 70 75 80Arg Pro Arg Asp
Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95Lys Gly Ser
Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110Thr
Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile 115 120
125Ile Ser Thr Leu Thr 13013133PRTArtificial SequenceIL2v 13Ala Pro
Ala Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His1 5 10 15Leu
Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25
30Asn Pro Lys Leu Thr Arg Met Leu Thr Ala Lys Phe Ala Met Pro Lys
35 40 45Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu
Lys 50 55 60Pro Leu Glu Glu Val Leu Asn Gly Ala Gln Ser Lys Asn Phe
His Leu65 70 75 80Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile
Val Leu Glu Leu 85 90 95Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr
Ala Asp Glu Thr Ala 100 105 110Thr Ile Val Glu Phe Leu Asn Arg Trp
Ile Thr Phe Ala Gln Ser Ile 115 120 125Ile Ser Thr Leu Thr
130145PRTArtificial SequenceCEA HCDR1 14Glu Phe Gly Met Asn1
51517PRTArtificial SequenceCEA HCDR2 15Trp Ile Asn Thr Lys Thr Gly
Glu Ala Thr Tyr Val Glu Glu Phe Lys1 5 10 15Gly1612PRTArtificial
SequenceCEA HCDR3 16Trp Asp Phe Ala Tyr Tyr Val Glu Ala Met Asp
Tyr1 5 101711PRTArtificial SequenceCEA LCDR1 17Lys Ala Ser Ala Ala
Val Gly Thr Tyr Val Ala1 5 10187PRTArtificial SequenceCEA LCDR2
18Ser Ala Ser Tyr Arg Lys Arg1 51910PRTArtificial SequenceCEA LCDR3
19His Gln Tyr Tyr Thr Tyr Pro Leu Phe Thr1 5 1020121PRTArtificial
SequenceCEA VH 20Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys
Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr
Thr Phe Thr Glu Phe 20 25 30Gly Met Asn Trp Val Arg Gln Ala Pro Gly
Gln Gly Leu Glu Trp Met 35 40 45Gly Trp Ile Asn Thr Lys Thr Gly Glu
Ala Thr Tyr Val Glu Glu Phe 50 55 60Lys Gly Arg Val Thr Phe Thr Thr
Asp Thr Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Arg Ser Leu
Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Trp Asp Phe
Ala Tyr Tyr Val Glu Ala Met Asp Tyr Trp Gly 100 105 110Gln Gly Thr
Thr Val Thr Val Ser Ser 115 12021108PRTArtificial SequenceCEA VL
21Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Ala Ala Val Gly Thr
Tyr 20 25 30Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile 35 40 45Tyr Ser Ala Ser Tyr Arg Lys Arg Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys His Gln
Tyr Tyr Thr Tyr Pro Leu 85 90 95Phe Thr Phe Gly Gln Gly Thr Lys Leu
Glu Ile Lys 100 10522598PRTArtificial SequenceCEA IL2v HC 22Gln Val
Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser
Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Glu Phe 20 25
30Gly Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45Gly Trp Ile Asn Thr Lys Thr Gly Glu Ala Thr Tyr Val Glu Glu
Phe 50 55 60Lys Gly Arg Val Thr Phe Thr Thr Asp Thr Ser Thr Ser Thr
Ala Tyr65 70 75 80Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala
Val Tyr Tyr Cys 85 90 95Ala Arg Trp Asp Phe Ala Tyr Tyr Val Glu Ala
Met Asp Tyr Trp Gly 100 105 110Gln Gly Thr Thr Val Thr Val Ser Ser
Ala Ser Thr Lys Gly Pro Ser 115 120 125Val Phe Pro Leu Ala Pro Ser
Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140Ala Leu Gly Cys Leu
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val145 150 155 160Ser Trp
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170
175Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val
180 185 190Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val
Asn His 195 200 205Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu
Pro Lys Ser Cys 210 215 220Asp Lys Thr His Thr Cys Pro Pro Cys Pro
Ala Pro Glu Ala Ala Gly225 230 235 240Gly Pro Ser Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255Ile Ser Arg Thr Pro
Glu Val Thr Cys Val Val Val Asp Val Ser His 260 265 270Glu Asp Pro
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280 285His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 290 295
300Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
Gly305 310 315 320Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
Gly Ala Pro Ile 325 330 335Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln Val 340 345 350Tyr Thr Leu Pro Pro Cys Arg Asp
Glu Leu Thr Lys Asn Gln Val Ser 355 360 365Leu Trp Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380Trp Glu Ser Asn
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro385 390 395 400Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 405 410
415Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
420 425 430His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser 435 440 445Pro Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
Gly Gly Gly Gly 450 455 460Ser Ala Pro Ala Ser Ser Ser Thr Lys Lys
Thr Gln Leu Gln Leu Glu465 470 475 480His Leu Leu Leu Asp Leu Gln
Met Ile Leu Asn Gly Ile Asn Asn Tyr 485 490 495Lys Asn Pro Lys Leu
Thr Arg Met Leu Thr Ala Lys Phe Ala Met Pro 500 505 510Lys Lys Ala
Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu 515 520 525Lys
Pro Leu Glu Glu Val Leu Asn Gly Ala Gln Ser Lys Asn Phe His 530 535
540Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu
Glu545 550 555 560Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr
Ala Asp Glu Thr 565 570 575Ala Thr Ile Val Glu Phe Leu Asn Arg Trp
Ile Thr Phe Ala Gln Ser 580 585 590Ile Ile Ser Thr Leu Thr
59523451PRTArtificial SequenceCEA HC 23Gln Val Gln Leu Val Gln Ser
Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys
Lys Ala Ser Gly Tyr Thr Phe Thr Glu Phe 20 25 30Gly Met Asn Trp Val
Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Trp Ile Asn
Thr Lys Thr Gly Glu Ala Thr Tyr Val Glu Glu Phe 50 55 60Lys Gly Arg
Val Thr Phe Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr65 70 75 80Met
Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90
95Ala Arg Trp Asp Phe Ala Tyr Tyr Val Glu Ala Met Asp Tyr Trp Gly
100 105 110Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly
Pro Ser 115 120 125Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser
Gly Gly Thr Ala 130 135 140Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe
Pro Glu Pro Val Thr Val145 150 155 160Ser Trp Asn Ser Gly Ala Leu
Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175Val Leu Gln Ser Ser
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190Pro Ser Ser
Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205Lys
Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 210 215
220Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala
Gly225 230 235 240Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
Asp Thr Leu Met 245 250 255Ile Ser Arg Thr Pro Glu Val Thr Cys Val
Val Val Asp Val Ser His 260 265 270Glu Asp Pro Glu Val Lys Phe Asn
Trp Tyr Val Asp Gly Val Glu Val 275 280 285His Asn Ala Lys Thr Lys
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 290 295 300Arg Val Val Ser
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly305 310 315 320Lys
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile 325 330
335Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
340 345 350Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
Val Ser 355 360 365Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu 370 375 380Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
Tyr Lys Thr Thr Pro Pro385 390 395 400Val Leu Asp Ser Asp Gly Ser
Phe Phe Leu Val Ser Lys Leu Thr Val
405 410 415Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
Val Met 420 425 430His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
Leu Ser Leu Ser 435 440 445Pro Gly Lys 45024215PRTArtificial
SequenceCEA LC 24Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser
Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Ala
Ala Val Gly Thr Tyr 20 25 30Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys
Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ser Ala Ser Tyr Arg Lys Arg Gly
Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr
Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr
Tyr Cys His Gln Tyr Tyr Thr Tyr Pro Leu 85 90 95Phe Thr Phe Gly Gln
Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala 100 105 110Ala Pro Ser
Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser 115 120 125Gly
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu 130 135
140Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn
Ser145 150 155 160Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
Thr Tyr Ser Leu 165 170 175Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp
Tyr Glu Lys His Lys Val 180 185 190Tyr Ala Cys Glu Val Thr His Gln
Gly Leu Ser Ser Pro Val Thr Lys 195 200 205Ser Phe Asn Arg Gly Glu
Cys 210 21525117PRTArtificial SequenceFAP VH 25Glu Val Gln Leu Leu
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Ala Met Ser
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Ala
Ile Ile Gly Ser Gly Ala Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Lys Gly Trp Phe Gly Gly Phe Asn Tyr Trp Gly Gln Gly Thr
Leu 100 105 110Val Thr Val Ser Ser 11526108PRTArtificial
SequenceFAP VL 26Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser
Leu Ser Pro Gly1 5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln
Ser Val Thr Ser Ser 20 25 30Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly
Gln Ala Pro Arg Leu Leu 35 40 45Ile Asn Val Gly Ser Arg Arg Ala Thr
Gly Ile Pro Asp Arg Phe Ser 50 55 60Gly Ser Gly Ser Gly Thr Asp Phe
Thr Leu Thr Ile Ser Arg Leu Glu65 70 75 80Pro Glu Asp Phe Ala Val
Tyr Tyr Cys Gln Gln Gly Ile Met Leu Pro 85 90 95Pro Thr Phe Gly Gln
Gly Thr Lys Val Glu Ile Lys 100 10527594PRTArtificial SequenceFAP
IL2v HC 27Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
Ser Ser Tyr 20 25 30Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu Trp Val 35 40 45Ser Ala Ile Ile Gly Ser Gly Ala Ser Thr Tyr
Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Lys Gly Trp Phe Gly Gly
Phe Asn Tyr Trp Gly Gln Gly Thr Leu 100 105 110Val Thr Val Ser Ser
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125Ala Pro Ser
Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys 130 135 140Leu
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser145 150
155 160Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
Ser 165 170 175Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
Ser Ser Ser 180 185 190Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn
His Lys Pro Ser Asn 195 200 205Thr Lys Val Asp Lys Lys Val Glu Pro
Lys Ser Cys Asp Lys Thr His 210 215 220Thr Cys Pro Pro Cys Pro Ala
Pro Glu Ala Ala Gly Gly Pro Ser Val225 230 235 240Phe Leu Phe Pro
Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250 255Pro Glu
Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu 260 265
270Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
275 280 285Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
Val Ser 290 295 300Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
Lys Glu Tyr Lys305 310 315 320Cys Lys Val Ser Asn Lys Ala Leu Gly
Ala Pro Ile Glu Lys Thr Ile 325 330 335Ser Lys Ala Lys Gly Gln Pro
Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350Pro Cys Arg Asp Glu
Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu 355 360 365Val Lys Gly
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375 380Gly
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser385 390
395 400Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
Arg 405 410 415Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
Glu Ala Leu 420 425 430His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
Ser Pro Gly Gly Gly 435 440 445Gly Gly Ser Gly Gly Gly Gly Ser Gly
Gly Gly Gly Ser Ala Pro Ala 450 455 460Ser Ser Ser Thr Lys Lys Thr
Gln Leu Gln Leu Glu His Leu Leu Leu465 470 475 480Asp Leu Gln Met
Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys 485 490 495Leu Thr
Arg Met Leu Thr Ala Lys Phe Ala Met Pro Lys Lys Ala Thr 500 505
510Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu
515 520 525Glu Val Leu Asn Gly Ala Gln Ser Lys Asn Phe His Leu Arg
Pro Arg 530 535 540Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu
Leu Lys Gly Ser545 550 555 560Glu Thr Thr Phe Met Cys Glu Tyr Ala
Asp Glu Thr Ala Thr Ile Val 565 570 575Glu Phe Leu Asn Arg Trp Ile
Thr Phe Ala Gln Ser Ile Ile Ser Thr 580 585 590Leu
Thr28447PRTArtificial SequenceFAP HC 28Glu Val Gln Leu Leu Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Ala Met Ser Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Ala Ile Ile
Gly Ser Gly Ala Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg
Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95Ala Lys Gly Trp Phe Gly Gly Phe Asn Tyr Trp Gly Gln Gly Thr Leu
100 105 110Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe
Pro Leu 115 120 125Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala
Ala Leu Gly Cys 130 135 140Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
Thr Val Ser Trp Asn Ser145 150 155 160Gly Ala Leu Thr Ser Gly Val
His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175Ser Gly Leu Tyr Ser
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180 185 190Leu Gly Thr
Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn 195 200 205Thr
Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His 210 215
220Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser
Val225 230 235 240Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
Ile Ser Arg Thr 245 250 255Pro Glu Val Thr Cys Val Val Val Asp Val
Ser His Glu Asp Pro Glu 260 265 270Val Lys Phe Asn Trp Tyr Val Asp
Gly Val Glu Val His Asn Ala Lys 275 280 285Thr Lys Pro Arg Glu Glu
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 290 295 300Val Leu Thr Val
Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys305 310 315 320Cys
Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys Thr Ile 325 330
335Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro
340 345 350Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Ser
Cys Ala 355 360 365Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
Trp Glu Ser Asn 370 375 380Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro Val Leu Asp Ser385 390 395 400Asp Gly Ser Phe Phe Leu Val
Ser Lys Leu Thr Val Asp Lys Ser Arg 405 410 415Trp Gln Gln Gly Asn
Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420 425 430His Asn His
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440
44529215PRTArtificial SequenceFAP LC 29Glu Ile Val Leu Thr Gln Ser
Pro Gly Thr Leu Ser Leu Ser Pro Gly1 5 10 15Glu Arg Ala Thr Leu Ser
Cys Arg Ala Ser Gln Ser Val Thr Ser Ser 20 25 30Tyr Leu Ala Trp Tyr
Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45Ile Asn Val Gly
Ser Arg Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60Gly Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu65 70 75 80Pro
Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Gly Ile Met Leu Pro 85 90
95Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala
100 105 110Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu
Lys Ser 115 120 125Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
Tyr Pro Arg Glu 130 135 140Ala Lys Val Gln Trp Lys Val Asp Asn Ala
Leu Gln Ser Gly Asn Ser145 150 155 160Gln Glu Ser Val Thr Glu Gln
Asp Ser Lys Asp Ser Thr Tyr Ser Leu 165 170 175Ser Ser Thr Leu Thr
Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val 180 185 190Tyr Ala Cys
Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys 195 200 205Ser
Phe Asn Arg Gly Glu Cys 210 21530225PRTHomo sapiens 30Asp Lys Thr
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly1 5 10 15Gly Pro
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30Ile
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40
45Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
50 55 60His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
Tyr65 70 75 80Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
Leu Asn Gly 85 90 95Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
Pro Ala Pro Ile 100 105 110Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln Val 115 120 125Tyr Thr Leu Pro Pro Ser Arg Asp
Glu Leu Thr Lys Asn Gln Val Ser 130 135 140Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu145 150 155 160Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185
190Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
195 200 205His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser 210 215 220Pro2253120PRTHomo sapiens 31Met Tyr Arg Met Gln
Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu1 5 10 15Val Thr Asn Ser
20325PRTArtificial SequenceCD3 HCDR1 32Thr Tyr Ala Met Asn1
53319PRTArtificial SequenceCD3 HCDR2 33Arg Ile Arg Ser Lys Tyr Asn
Asn Tyr Ala Thr Tyr Tyr Ala Asp Ser1 5 10 15Val Lys
Gly3414PRTArtificial SequenceCD3 HCDR3 34His Gly Asn Phe Gly Asn
Ser Tyr Val Ser Trp Phe Ala Tyr1 5 103514PRTArtificial SequenceCD3
LCDR1 35Gly Ser Ser Thr Gly Ala Val Thr Thr Ser Asn Tyr Ala Asn1 5
10367PRTArtificial SequenceCD3 LCDR2 36Gly Thr Asn Lys Arg Ala Pro1
5379PRTArtificial SequenceCD3 LCDR3 37Ala Leu Trp Tyr Ser Asn Leu
Trp Val1 538125PRTArtificial SequenceCD3 VH 38Glu Val Gln Leu Leu
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr 20 25 30Ala Met Asn
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Arg
Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60Ser
Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr65 70 75
80Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
85 90 95Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp
Phe 100 105 110Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 12539109PRTArtificial SequenceCD3 VL 39Gln Ala Val Val Thr
Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly1 5 10 15Thr Val Thr Leu
Thr Cys Gly Ser Ser Thr Gly Ala Val Thr Thr Ser 20 25 30Asn Tyr Ala
Asn Trp Val Gln Glu Lys Pro Gly Gln Ala Phe Arg Gly 35 40 45Leu Ile
Gly Gly Thr Asn Lys Arg Ala Pro Gly Thr Pro Ala Arg Phe 50 55 60Ser
Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly Ala65 70 75
80Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn
85 90 95Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100
10540215PRTArtificial SequenceCEA CD3 bsAb LC(CEA) 40Asp Ile Gln
Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg
Val Thr Ile Thr Cys Lys Ala Ser Ala Ala Val Gly Thr Tyr 20 25 30Val
Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40
45Tyr Ser Ala Ser Tyr Arg Lys Arg Gly Val Pro Ser Arg Phe Ser Gly
50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln
Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys His Gln Tyr Tyr Thr
Tyr Pro Leu 85 90 95Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
Arg Thr Val Ala 100 105 110Ala Pro Ser Val Phe Ile Phe Pro Pro Ser
Asp Glu Gln Leu Lys Ser
115 120 125Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro
Arg Glu 130 135 140Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln
Ser Gly Asn Ser145 150 155 160Gln Glu Ser Val Thr Glu Gln Asp Ser
Lys Asp Ser Thr Tyr Ser Leu 165 170 175Ser Ser Thr Leu Thr Leu Ser
Lys Ala Asp Tyr Glu Lys His Lys Val 180 185 190Tyr Ala Cys Glu Val
Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys 195 200 205Ser Phe Asn
Arg Gly Glu Cys 210 21541214PRTArtificial SequenceCEA CD3 bsAb
LC(CD3) 41Gln Ala Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro
Gly Gly1 5 10 15Thr Val Thr Leu Thr Cys Gly Ser Ser Thr Gly Ala Val
Thr Thr Ser 20 25 30Asn Tyr Ala Asn Trp Val Gln Glu Lys Pro Gly Gln
Ala Phe Arg Gly 35 40 45Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly
Thr Pro Ala Arg Phe 50 55 60Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala
Leu Thr Leu Ser Gly Ala65 70 75 80Gln Pro Glu Asp Glu Ala Glu Tyr
Tyr Cys Ala Leu Trp Tyr Ser Asn 85 90 95Leu Trp Val Phe Gly Gly Gly
Thr Lys Leu Thr Val Leu Ser Ser Ala 100 105 110Ser Thr Lys Gly Pro
Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser 115 120 125Thr Ser Gly
Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe 130 135 140Pro
Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly145 150
155 160Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
Leu 165 170 175Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr
Gln Thr Tyr 180 185 190Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr
Lys Val Asp Lys Lys 195 200 205Val Glu Pro Lys Ser Cys
21042694PRTArtificial SequenceCEA CD3 bsAB HC(CEA-CD3-Fc) 42Gln Val
Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser
Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Glu Phe 20 25
30Gly Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45Gly Trp Ile Asn Thr Lys Thr Gly Glu Ala Thr Tyr Val Glu Glu
Phe 50 55 60Lys Gly Arg Val Thr Phe Thr Thr Asp Thr Ser Thr Ser Thr
Ala Tyr65 70 75 80Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala
Val Tyr Tyr Cys 85 90 95Ala Arg Trp Asp Phe Ala Tyr Tyr Val Glu Ala
Met Asp Tyr Trp Gly 100 105 110Gln Gly Thr Thr Val Thr Val Ser Ser
Ala Ser Thr Lys Gly Pro Ser 115 120 125Val Phe Pro Leu Ala Pro Ser
Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140Ala Leu Gly Cys Leu
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val145 150 155 160Ser Trp
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170
175Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val
180 185 190Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val
Asn His 195 200 205Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu
Pro Lys Ser Cys 210 215 220Asp Gly Gly Gly Gly Ser Gly Gly Gly Gly
Ser Glu Val Gln Leu Leu225 230 235 240Glu Ser Gly Gly Gly Leu Val
Gln Pro Gly Gly Ser Leu Arg Leu Ser 245 250 255Cys Ala Ala Ser Gly
Phe Thr Phe Ser Thr Tyr Ala Met Asn Trp Val 260 265 270Arg Gln Ala
Pro Gly Lys Gly Leu Glu Trp Val Ser Arg Ile Arg Ser 275 280 285Lys
Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg 290 295
300Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr Leu Tyr Leu Gln
Met305 310 315 320Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr
Cys Val Arg His 325 330 335Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp
Phe Ala Tyr Trp Gly Gln 340 345 350Gly Thr Leu Val Thr Val Ser Ser
Ala Ser Val Ala Ala Pro Ser Val 355 360 365Phe Ile Phe Pro Pro Ser
Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser 370 375 380Val Val Cys Leu
Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln385 390 395 400Trp
Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val 405 410
415Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu
420 425 430Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala
Cys Glu 435 440 445Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys
Ser Phe Asn Arg 450 455 460Gly Glu Cys Asp Lys Thr His Thr Cys Pro
Pro Cys Pro Ala Pro Glu465 470 475 480Ala Ala Gly Gly Pro Ser Val
Phe Leu Phe Pro Pro Lys Pro Lys Asp 485 490 495Thr Leu Met Ile Ser
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 500 505 510Val Ser His
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 515 520 525Val
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn 530 535
540Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
Trp545 550 555 560Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
Lys Ala Leu Gly 565 570 575Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala
Lys Gly Gln Pro Arg Glu 580 585 590Pro Gln Val Tyr Thr Leu Pro Pro
Cys Arg Asp Glu Leu Thr Lys Asn 595 600 605Gln Val Ser Leu Trp Cys
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 610 615 620Ala Val Glu Trp
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr625 630 635 640Thr
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 645 650
655Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
660 665 670Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
Ser Leu 675 680 685Ser Leu Ser Pro Gly Lys 69043451PRTArtificial
SequenceCEA CD3 bsAB HC(CEA-Fc) 43Gln Val Gln Leu Val Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys
Ala Ser Gly Tyr Thr Phe Thr Glu Phe 20 25 30Gly Met Asn Trp Val Arg
Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Trp Ile Asn Thr
Lys Thr Gly Glu Ala Thr Tyr Val Glu Glu Phe 50 55 60Lys Gly Arg Val
Thr Phe Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu
Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala
Arg Trp Asp Phe Ala Tyr Tyr Val Glu Ala Met Asp Tyr Trp Gly 100 105
110Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser
115 120 125Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly
Thr Ala 130 135 140Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu
Pro Val Thr Val145 150 155 160Ser Trp Asn Ser Gly Ala Leu Thr Ser
Gly Val His Thr Phe Pro Ala 165 170 175Val Leu Gln Ser Ser Gly Leu
Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190Pro Ser Ser Ser Leu
Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205Lys Pro Ser
Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 210 215 220Asp
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly225 230
235 240Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
Met 245 250 255Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
Val Ser His 260 265 270Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
Asp Gly Val Glu Val 275 280 285His Asn Ala Lys Thr Lys Pro Arg Glu
Glu Gln Tyr Asn Ser Thr Tyr 290 295 300Arg Val Val Ser Val Leu Thr
Val Leu His Gln Asp Trp Leu Asn Gly305 310 315 320Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile 325 330 335Glu Lys
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345
350Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser
355 360 365Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
Val Glu 370 375 380Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro385 390 395 400Val Leu Asp Ser Asp Gly Ser Phe Phe
Leu Val Ser Lys Leu Thr Val 405 410 415Asp Lys Ser Arg Trp Gln Gln
Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430His Glu Ala Leu His
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445Pro Gly Lys
45044672PRTArtificial SequenceCD20 VH-CH1(EE)-CD3 VL-CH1-Fc (knob,
P329G LALA) 44Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys
Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala
Phe Ser Tyr Ser 20 25 30Trp Ile Asn Trp Val Arg Gln Ala Pro Gly Gln
Gly Leu Glu Trp Met 35 40 45Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr
Asp Tyr Asn Gly Lys Phe 50 55 60Lys Gly Arg Val Thr Ile Thr Ala Asp
Lys Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg
Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Asn Val Phe Asp
Gly Tyr Trp Leu Val Tyr Trp Gly Gln Gly 100 105 110Thr Leu Val Thr
Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125Pro Leu
Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135
140Gly Cys Leu Val Glu Asp Tyr Phe Pro Glu Pro Val Thr Val Ser
Trp145 150 155 160Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe
Pro Ala Val Leu 165 170 175Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser
Val Val Thr Val Pro Ser 180 185 190Ser Ser Leu Gly Thr Gln Thr Tyr
Ile Cys Asn Val Asn His Lys Pro 195 200 205Ser Asn Thr Lys Val Asp
Glu Lys Val Glu Pro Lys Ser Cys Asp Gly 210 215 220Gly Gly Gly Ser
Gly Gly Gly Gly Ser Gln Ala Val Val Thr Gln Glu225 230 235 240Pro
Ser Leu Thr Val Ser Pro Gly Gly Thr Val Thr Leu Thr Cys Gly 245 250
255Ser Ser Thr Gly Ala Val Thr Thr Ser Asn Tyr Ala Asn Trp Val Gln
260 265 270Glu Lys Pro Gly Gln Ala Phe Arg Gly Leu Ile Gly Gly Thr
Asn Lys 275 280 285Arg Ala Pro Gly Thr Pro Ala Arg Phe Ser Gly Ser
Leu Leu Gly Gly 290 295 300Lys Ala Ala Leu Thr Leu Ser Gly Ala Gln
Pro Glu Asp Glu Ala Glu305 310 315 320Tyr Tyr Cys Ala Leu Trp Tyr
Ser Asn Leu Trp Val Phe Gly Gly Gly 325 330 335Thr Lys Leu Thr Val
Leu Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 340 345 350Phe Pro Leu
Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 355 360 365Leu
Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 370 375
380Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
Val385 390 395 400Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val
Val Thr Val Pro 405 410 415Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile
Cys Asn Val Asn His Lys 420 425 430Pro Ser Asn Thr Lys Val Asp Lys
Lys Val Glu Pro Lys Ser Cys Asp 435 440 445Lys Thr His Thr Cys Pro
Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly 450 455 460Pro Ser Val Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile465 470 475 480Ser
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 485 490
495Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
500 505 510Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
Tyr Arg 515 520 525Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
Leu Asn Gly Lys 530 535 540Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
Leu Gly Ala Pro Ile Glu545 550 555 560Lys Thr Ile Ser Lys Ala Lys
Gly Gln Pro Arg Glu Pro Gln Val Tyr 565 570 575Thr Leu Pro Pro Cys
Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu 580 585 590Trp Cys Leu
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 595 600 605Glu
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 610 615
620Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
Asp625 630 635 640Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
Ser Val Met His 645 650 655Glu Ala Leu His Asn His Tyr Thr Gln Lys
Ser Leu Ser Leu Ser Pro 660 665 67045447PRTArtificial SequenceCD20
VH-CH1(EE)-Fc (hole, P329G LALA) 45Gln Val Gln Leu Val Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys
Ala Ser Gly Tyr Ala Phe Ser Tyr Ser 20 25 30Trp Ile Asn Trp Val Arg
Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Arg Ile Phe Pro
Gly Asp Gly Asp Thr Asp Tyr Asn Gly Lys Phe 50 55 60Lys Gly Arg Val
Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu
Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala
Arg Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr Trp Gly Gln Gly 100 105
110Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe
115 120 125Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala
Ala Leu 130 135 140Gly Cys Leu Val Glu Asp Tyr Phe Pro Glu Pro Val
Thr Val Ser Trp145 150 155 160Asn Ser Gly Ala Leu Thr Ser Gly Val
His Thr Phe Pro Ala Val Leu 165 170 175Gln Ser Ser Gly Leu Tyr Ser
Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190Ser Ser Leu Gly Thr
Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205Ser Asn Thr
Lys Val Asp Glu Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220Thr
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro225 230
235 240Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
Ser 245 250 255Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
His Glu Asp 260 265 270Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
Val Glu Val His Asn 275 280 285Ala Lys Thr Lys Pro Arg Glu Glu Gln
Tyr Asn Ser Thr Tyr Arg Val 290 295 300Val Ser Val Leu Thr Val Leu
His Gln Asp Trp Leu Asn Gly Lys Glu305
310 315 320Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile
Glu Lys 325 330 335Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
Gln Val Cys Thr 340 345 350Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys
Asn Gln Val Ser Leu Ser 355 360 365Cys Ala Val Lys Gly Phe Tyr Pro
Ser Asp Ile Ala Val Glu Trp Glu 370 375 380Ser Asn Gly Gln Pro Glu
Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu385 390 395 400Asp Ser Asp
Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys 405 410 415Ser
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425
430Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435
440 44546219PRTArtificial SequenceCD20 VL-CL(RK) 46Asp Ile Val Met
Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Pro Gly1 5 10 15Glu Pro Ala
Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser 20 25 30Asn Gly
Ile Thr Tyr Leu Tyr Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45Pro
Gln Leu Leu Ile Tyr Gln Met Ser Asn Leu Val Ser Gly Val Pro 50 55
60Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile65
70 75 80Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ala Gln
Asn 85 90 95Leu Glu Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu
Ile Lys 100 105 110Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro
Pro Ser Asp Arg 115 120 125Lys Leu Lys Ser Gly Thr Ala Ser Val Val
Cys Leu Leu Asn Asn Phe 130 135 140Tyr Pro Arg Glu Ala Lys Val Gln
Trp Lys Val Asp Asn Ala Leu Gln145 150 155 160Ser Gly Asn Ser Gln
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 165 170 175Thr Tyr Ser
Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 180 185 190Lys
His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 195 200
205Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210
21547232PRTArtificial SequenceCD3 VH-CL 47Glu Val Gln Leu Leu Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr 20 25 30Ala Met Asn Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Arg Ile
Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60Ser Val
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr65 70 75
80Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
85 90 95Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp
Phe 100 105 110Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
Ala Ser Val 115 120 125Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser
Asp Glu Gln Leu Lys 130 135 140Ser Gly Thr Ala Ser Val Val Cys Leu
Leu Asn Asn Phe Tyr Pro Arg145 150 155 160Glu Ala Lys Val Gln Trp
Lys Val Asp Asn Ala Leu Gln Ser Gly Asn 165 170 175Ser Gln Glu Ser
Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser 180 185 190Leu Ser
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys 195 200
205Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr
210 215 220Lys Ser Phe Asn Arg Gly Glu Cys225 230485PRTArtificial
SequenceCD19 HCDR1 48Asp Tyr Ile Met His1 54917PRTArtificial
SequenceCD19 HCDR2 49Tyr Ile Asn Pro Tyr Asn Asp Gly Ser Lys Tyr
Thr Glu Lys Phe Gln1 5 10 15Gly5012PRTArtificial SequenceCD19 HCDR3
50Gly Thr Tyr Tyr Tyr Gly Ser Ala Leu Phe Asp Tyr1 5
105116PRTArtificial SequenceCD19 LCDR1 51Lys Ser Ser Gln Ser Leu
Glu Asn Pro Asn Gly Asn Thr Tyr Leu Asn1 5 10 15527PRTArtificial
SequenceCD19 LCDR2 52Arg Val Ser Lys Arg Phe Ser1 5539PRTArtificial
SequenceCD19 LCDR3 53Leu Gln Leu Thr His Val Pro Tyr Thr1
554121PRTArtificial SequenceCD19 VH 54Gln Val Gln Leu Val Gln Ser
Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys
Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30Ile Met His Trp Val
Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Tyr Ile Asn
Pro Tyr Asn Asp Gly Ser Lys Tyr Thr Glu Lys Phe 50 55 60Gln Gly Arg
Val Thr Met Thr Ser Asp Thr Ser Ile Ser Thr Ala Tyr65 70 75 80Met
Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90
95Ala Arg Gly Thr Tyr Tyr Tyr Gly Ser Ala Leu Phe Asp Tyr Trp Gly
100 105 110Gln Gly Thr Thr Val Thr Val Ser Ser 115
12055112PRTArtificial SequenceCD19 VL 55Asp Ile Val Met Thr Gln Thr
Pro Leu Ser Leu Ser Val Thr Pro Gly1 5 10 15Gln Pro Ala Ser Ile Ser
Cys Lys Ser Ser Gln Ser Leu Glu Asn Pro 20 25 30Asn Gly Asn Thr Tyr
Leu Asn Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45Pro Gln Leu Leu
Ile Tyr Arg Val Ser Lys Arg Phe Ser Gly Val Pro 50 55 60Asp Arg Phe
Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile65 70 75 80Ser
Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Leu Gln Leu 85 90
95Thr His Val Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105 11056449PRTArtificial SequenceCD19 VH-CH1(EE)-Fc(hole,
P329G LALA) 56Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys
Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr
Phe Thr Asp Tyr 20 25 30Ile Met His Trp Val Arg Gln Ala Pro Gly Gln
Gly Leu Glu Trp Met 35 40 45Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Ser
Lys Tyr Thr Glu Lys Phe 50 55 60Gln Gly Arg Val Thr Met Thr Ser Asp
Thr Ser Ile Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Arg Leu Arg
Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Thr Tyr Tyr
Tyr Gly Ser Ala Leu Phe Asp Tyr Trp Gly 100 105 110Gln Gly Thr Thr
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125Val Phe
Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135
140Ala Leu Gly Cys Leu Val Glu Asp Tyr Phe Pro Glu Pro Val Thr
Val145 150 155 160Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His
Thr Phe Pro Ala 165 170 175Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu
Ser Ser Val Val Thr Val 180 185 190Pro Ser Ser Ser Leu Gly Thr Gln
Thr Tyr Ile Cys Asn Val Asn His 195 200 205Lys Pro Ser Asn Thr Lys
Val Asp Glu Lys Val Glu Pro Lys Ser Cys 210 215 220Asp Lys Thr His
Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly225 230 235 240Gly
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250
255Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
260 265 270Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
Glu Val 275 280 285His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
Asn Ser Thr Tyr 290 295 300Arg Val Val Ser Val Leu Thr Val Leu His
Gln Asp Trp Leu Asn Gly305 310 315 320Lys Glu Tyr Lys Cys Lys Val
Ser Asn Lys Ala Leu Gly Ala Pro Ile 325 330 335Glu Lys Thr Ile Ser
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345 350Cys Thr Leu
Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 355 360 365Leu
Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375
380Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
Pro385 390 395 400Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser
Lys Leu Thr Val 405 410 415Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
Phe Ser Cys Ser Val Met 420 425 430His Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445Pro57674PRTArtificial
SequenceCD19 VH-CH1(EE)-CD3 VL-CH1-Fc(knob, P329G LALA) 57Gln Val
Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser
Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25
30Ile Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Ser Lys Tyr Thr Glu Lys
Phe 50 55 60Gln Gly Arg Val Thr Met Thr Ser Asp Thr Ser Ile Ser Thr
Ala Tyr65 70 75 80Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala
Val Tyr Tyr Cys 85 90 95Ala Arg Gly Thr Tyr Tyr Tyr Gly Ser Ala Leu
Phe Asp Tyr Trp Gly 100 105 110Gln Gly Thr Thr Val Thr Val Ser Ser
Ala Ser Thr Lys Gly Pro Ser 115 120 125Val Phe Pro Leu Ala Pro Ser
Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140Ala Leu Gly Cys Leu
Val Glu Asp Tyr Phe Pro Glu Pro Val Thr Val145 150 155 160Ser Trp
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170
175Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val
180 185 190Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val
Asn His 195 200 205Lys Pro Ser Asn Thr Lys Val Asp Glu Lys Val Glu
Pro Lys Ser Cys 210 215 220Asp Gly Gly Gly Gly Ser Gly Gly Gly Gly
Ser Gln Ala Val Val Thr225 230 235 240Gln Glu Pro Ser Leu Thr Val
Ser Pro Gly Gly Thr Val Thr Leu Thr 245 250 255Cys Gly Ser Ser Thr
Gly Ala Val Thr Thr Ser Asn Tyr Ala Asn Trp 260 265 270Val Gln Glu
Lys Pro Gly Gln Ala Phe Arg Gly Leu Ile Gly Gly Thr 275 280 285Asn
Lys Arg Ala Pro Gly Thr Pro Ala Arg Phe Ser Gly Ser Leu Leu 290 295
300Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly Ala Gln Pro Glu Asp
Glu305 310 315 320Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn Leu
Trp Val Phe Gly 325 330 335Gly Gly Thr Lys Leu Thr Val Leu Ser Ser
Ala Ser Thr Lys Gly Pro 340 345 350Ser Val Phe Pro Leu Ala Pro Ser
Ser Lys Ser Thr Ser Gly Gly Thr 355 360 365Ala Ala Leu Gly Cys Leu
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 370 375 380Val Ser Trp Asn
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro385 390 395 400Ala
Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 405 410
415Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn
420 425 430His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro
Lys Ser 435 440 445Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
Pro Glu Ala Ala 450 455 460Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
Lys Pro Lys Asp Thr Leu465 470 475 480Met Ile Ser Arg Thr Pro Glu
Val Thr Cys Val Val Val Asp Val Ser 485 490 495His Glu Asp Pro Glu
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 500 505 510Val His Asn
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 515 520 525Tyr
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 530 535
540Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala
Pro545 550 555 560Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
Arg Glu Pro Gln 565 570 575Val Tyr Thr Leu Pro Pro Cys Arg Asp Glu
Leu Thr Lys Asn Gln Val 580 585 590Ser Leu Trp Cys Leu Val Lys Gly
Phe Tyr Pro Ser Asp Ile Ala Val 595 600 605Glu Trp Glu Ser Asn Gly
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 610 615 620Pro Val Leu Asp
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr625 630 635 640Val
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 645 650
655Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
660 665 670Ser Pro58219PRTArtificial SequenceCD19 VL-CL(RK) 58Asp
Ile Val Met Thr Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly1 5 10
15Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Glu Asn Pro
20 25 30Asn Gly Asn Thr Tyr Leu Asn Trp Tyr Leu Gln Lys Pro Gly Gln
Ser 35 40 45Pro Gln Leu Leu Ile Tyr Arg Val Ser Lys Arg Phe Ser Gly
Val Pro 50 55 60Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
Leu Lys Ile65 70 75 80Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr
Tyr Cys Leu Gln Leu 85 90 95Thr His Val Pro Tyr Thr Phe Gly Gln Gly
Thr Lys Leu Glu Ile Lys 100 105 110Arg Thr Val Ala Ala Pro Ser Val
Phe Ile Phe Pro Pro Ser Asp Arg 115 120 125Lys Leu Lys Ser Gly Thr
Ala Ser Val Val Cys Leu Leu Asn Asn Phe 130 135 140Tyr Pro Arg Glu
Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln145 150 155 160Ser
Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 165 170
175Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
180 185 190Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu
Ser Ser 195 200 205Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210
215595PRTArtificial SequenceCD19 HCDR1 59Asp Tyr Ile Met His1
56017PRTArtificial SequenceCD19 HCDR2 60Tyr Ile Asn Pro Tyr Asn Asp
Gly Ser Lys Tyr Thr Glu Lys Phe Gln1 5 10 15Gly6112PRTArtificial
SequenceCD19 HCDR3 61Gly Thr Tyr Tyr Tyr Gly Pro Gln Leu Phe Asp
Tyr1 5 106216PRTArtificial SequenceCD19 LCDR1 62Lys Ser Ser Gln Ser
Leu Glu Thr Ser Thr Gly Thr Thr Tyr Leu Asn1 5 10
15637PRTArtificial SequenceCD19 LCDR2 63Arg Val Ser Lys Arg Phe
Ser1 5649PRTArtificial SequenceCD19 LCDR3 64Leu Gln Leu Leu Glu Asp
Pro Tyr Thr1 565121PRTArtificial SequenceCD19 VH 65Gln Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys
Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30Ile Met
His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly
Tyr Ile Asn Pro Tyr Asn Asp Gly Ser Lys Tyr Thr Glu Lys Phe 50 55
60Gln Gly Arg Val Thr Met Thr Ser Asp Thr Ser Ile Ser Thr Ala Tyr65
70 75 80Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr
Tyr Cys 85 90 95Ala Arg Gly Thr Tyr Tyr Tyr Gly Pro Gln Leu Phe Asp
Tyr Trp Gly 100 105 110Gln Gly Thr Thr Val Thr Val Ser Ser 115
12066112PRTArtificial SequenceCD19 VL 66Asp Ile Val Met Thr Gln Thr
Pro Leu Ser Leu Ser Val Thr Pro Gly1 5 10 15Gln Pro Ala Ser Ile Ser
Cys Lys Ser Ser Gln Ser Leu Glu Thr Ser 20 25 30Thr Gly Thr Thr Tyr
Leu Asn Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45Pro Gln Leu Leu
Ile Tyr Arg Val Ser Lys Arg Phe Ser Gly Val Pro 50 55 60Asp Arg Phe
Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile65 70 75 80Ser
Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Leu Gln Leu 85 90
95Leu Glu Asp Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105 110675PRTArtificial SequenceCD19 HCDR1 67Asp Tyr Ile Met
His1 56817PRTArtificial SequenceCD19 HCDR2 68Tyr Ile Asn Pro Tyr
Asn Asp Gly Ser Lys Tyr Thr Glu Lys Phe Gln1 5 10
15Gly6912PRTArtificial SequenceCD19 HCDR3 69Gly Thr Tyr Tyr Tyr Gly
Ser Ala Leu Phe Asp Tyr1 5 107016PRTArtificial SequenceCD19 LCDR1
70Lys Ser Ser Gln Ser Leu Glu Ser Ser Thr Gly Asn Thr Tyr Leu Asn1
5 10 15717PRTArtificial SequenceCD19 LCDR2 71Arg Val Ser Lys Arg
Phe Ser1 5729PRTArtificial SequenceCD19 LCDR3 72Leu Gln Leu Ile Asp
Tyr Pro Val Thr1 573121PRTArtificial SequenceCD19 VH 73Gln Val Gln
Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val
Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30Ile
Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40
45Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Ser Lys Tyr Thr Glu Lys Phe
50 55 60Gln Gly Arg Val Thr Met Thr Ser Asp Thr Ser Ile Ser Thr Ala
Tyr65 70 75 80Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val
Tyr Tyr Cys 85 90 95Ala Arg Gly Thr Tyr Tyr Tyr Gly Ser Ala Leu Phe
Asp Tyr Trp Gly 100 105 110Gln Gly Thr Thr Val Thr Val Ser Ser 115
12074112PRTArtificial SequenceCD19 VL 74Asp Ile Val Met Thr Gln Thr
Pro Leu Ser Leu Ser Val Thr Pro Gly1 5 10 15Gln Pro Ala Ser Ile Ser
Cys Lys Ser Ser Gln Ser Leu Glu Ser Ser 20 25 30Thr Gly Asn Thr Tyr
Leu Asn Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45Pro Gln Leu Leu
Ile Tyr Arg Val Ser Lys Arg Phe Ser Gly Val Pro 50 55 60Asp Arg Phe
Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile65 70 75 80Ser
Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Leu Gln Leu 85 90
95Ile Asp Tyr Pro Val Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105 110755PRTArtificial SequenceCD19 HCDR1 75Asp Tyr Ile Met
His1 57617PRTArtificial SequenceCD19 HCDR2 76Tyr Ile Asn Pro Tyr
Asn Asp Gly Ser Lys Tyr Thr Glu Lys Phe Gln1 5 10
15Gly7712PRTArtificial SequenceCD19 HCDR3 77Gly Thr Tyr Tyr Tyr Gly
Ser Glu Leu Phe Asp Tyr1 5 107816PRTArtificial SequenceCD19 LCDR1
78Lys Ser Ser Gln Ser Leu Glu Thr Ser Thr Gly Asn Thr Tyr Leu Asn1
5 10 15797PRTArtificial SequenceCD19 LCDR2 79Arg Val Ser Lys Arg
Phe Ser1 5809PRTArtificial SequenceCD19 LCDR3 80Leu Gln Ala Thr His
Ile Pro Tyr Thr1 581121PRTArtificial SequenceCD19 VH 81Gln Val Gln
Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val
Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30Ile
Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40
45Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Ser Lys Tyr Thr Glu Lys Phe
50 55 60Gln Gly Arg Val Thr Met Thr Ser Asp Thr Ser Ile Ser Thr Ala
Tyr65 70 75 80Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val
Tyr Tyr Cys 85 90 95Ala Arg Gly Thr Tyr Tyr Tyr Gly Ser Glu Leu Phe
Asp Tyr Trp Gly 100 105 110Gln Gly Thr Thr Val Thr Val Ser Ser 115
12082112PRTArtificial SequenceCD19 VL 82Asp Ile Val Met Thr Gln Thr
Pro Leu Ser Leu Ser Val Thr Pro Gly1 5 10 15Gln Pro Ala Ser Ile Ser
Cys Lys Ser Ser Gln Ser Leu Glu Thr Ser 20 25 30Thr Gly Asn Thr Tyr
Leu Asn Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45Pro Gln Leu Leu
Ile Tyr Arg Val Ser Lys Arg Phe Ser Gly Val Pro 50 55 60Asp Arg Phe
Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile65 70 75 80Ser
Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Leu Gln Ala 85 90
95Thr His Ile Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105 110835PRTArtificial SequenceCD19 HCDR1 83Asp Tyr Ile Thr
His1 58417PRTArtificial SequenceCD19 HCDR2 84Tyr Ile Asn Pro Tyr
Asn Asp Gly Ser Lys Tyr Thr Glu Lys Phe Gln1 5 10
15Gly8512PRTArtificial SequenceCD19 HCDR3 85Gly Thr Tyr Tyr Tyr Gly
Pro Asp Leu Phe Asp Tyr1 5 108616PRTArtificial SequenceCD19 LCDR1
86Lys Ser Ser Gln Ser Leu Glu Thr Ser Thr Gly Asn Thr Tyr Leu Asn1
5 10 15877PRTArtificial SequenceCD19 LCDR2 87Arg Val Ser Lys Arg
Phe Ser1 5889PRTArtificial SequenceCD19 LCDR3 88Leu Gln Leu Thr His
Val Pro Tyr Thr1 589121PRTArtificial SequenceCD19 VH 89Gln Val Gln
Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val
Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30Ile
Thr His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40
45Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Ser Lys Tyr Thr Glu Lys Phe
50 55 60Gln Gly Arg Val Thr Met Thr Ser Asp Thr Ser Ile Ser Thr Ala
Tyr65 70 75 80Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val
Tyr Tyr Cys 85 90 95Ala Arg Gly Thr Tyr Tyr Tyr Gly Pro Asp Leu Phe
Asp Tyr Trp Gly 100 105 110Gln Gly Thr Thr Val Thr Val Ser Ser 115
12090112PRTArtificial SequenceCD19 VLmisc_feature(107)..(107)Xaa
can be any naturally occurring amino acid 90Asp Ile Val Met Thr Gln
Thr Pro Leu Ser Leu Ser Val Thr Pro Gly1 5 10 15Gln Pro Ala Ser Ile
Ser Cys Lys Ser Ser Gln Ser Leu Glu Thr Ser 20 25 30Thr Gly Asn Thr
Tyr Leu Asn Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45Pro Gln Leu
Leu Ile Tyr Arg Val Ser Lys Arg Phe Ser Gly Val Pro 50 55 60Asp Arg
Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile65 70 75
80Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Leu Gln Leu
85 90 95Thr His Val Pro Tyr Thr Phe Gly Gln Gly Xaa Lys Leu Glu Ile
Lys 100 105 110915PRTArtificial SequenceCD19 HCDR1 91Asp Tyr Ile
Met His1 59217PRTArtificial SequenceCD19 HCDR2 92Tyr Ile Asn Pro
Tyr Asn Asp Gly Ser Lys Tyr Thr Glu Lys Phe Gln1 5 10
15Gly9312PRTArtificial SequenceCD19 HCDR3 93Gly Thr Tyr Tyr Tyr Gly
Ser Ala Leu Phe Asp Tyr1 5 109416PRTArtificial SequenceCD19 LCDR1
94Lys Ser Ser Gln Ser Leu Glu Thr Ser Thr Gly Asn Thr Tyr Leu Asn1
5 10 15957PRTArtificial SequenceCD19 LCDR2 95Arg Val Ser Lys Arg
Phe Ser1 5969PRTArtificial SequenceCD19 LCDR3 96Leu Gln Pro Gly His
Tyr Pro Gly Thr1 597121PRTArtificial SequenceCD19 VH 97Gln Val Gln
Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val
Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30Ile
Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40
45Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Ser Lys Tyr Thr Glu Lys Phe
50 55 60Gln Gly Arg Val Thr Met Thr Ser Asp Thr Ser Ile Ser Thr Ala
Tyr65 70 75 80Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val
Tyr Tyr Cys 85 90 95Ala Arg Gly Thr Tyr Tyr Tyr Gly Ser Ala Leu Phe
Asp Tyr Trp Gly 100 105 110Gln Gly Thr Thr Val Thr Val Ser Ser 115
12098112PRTArtificial SequenceCD19 VL 98Asp Ile Val Met Thr Gln Thr
Pro Leu Ser Leu Ser Val Thr Pro Gly1 5 10 15Gln Pro Ala Ser Ile Ser
Cys Lys Ser Ser Gln Ser Leu Glu Thr Ser 20 25 30Thr Gly Asn Thr Tyr
Leu Asn Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45Pro Gln Leu Leu
Ile Tyr Arg Val Ser Lys Arg Phe Ser Gly Val Pro 50 55 60Asp Arg Phe
Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile65 70 75 80Ser
Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Leu Gln Pro 85 90
95Gly His Tyr Pro Gly Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105 110995PRTArtificial SequenceCD19 HCDR1 99Asp Tyr Ile Met
His1 510017PRTArtificial SequenceCD19 HCDR2 100Tyr Ile Asn Pro Tyr
Asn Asp Gly Ser Lys Tyr Thr Glu Lys Phe Gln1 5 10
15Gly10112PRTArtificial SequenceCD19 HCDR3 101Gly Thr Tyr Tyr Tyr
Gly Pro Gln Leu Phe Asp Tyr1 5 1010216PRTArtificial SequenceCD19
LCDR1 102Lys Ser Ser Gln Ser Leu Glu Thr Ser Thr Gly Asn Thr Tyr
Leu Asn1 5 10 151037PRTArtificial SequenceCD19 LCDR2 103Arg Val Ser
Lys Arg Phe Ser1 51049PRTArtificial SequenceCD19 LCDR3 104Leu Gln
Leu Asp Ser Tyr Pro Asn Thr1 5105121PRTArtificial SequenceCD19 VH
105Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1
5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp
Tyr 20 25 30Ile Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu
Trp Met 35 40 45Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Ser Lys Tyr Thr
Glu Lys Phe 50 55 60Gln Gly Arg Val Thr Met Thr Ser Asp Thr Ser Ile
Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Arg Leu Arg Ser Asp Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Thr Tyr Tyr Tyr Gly Pro
Gln Leu Phe Asp Tyr Trp Gly 100 105 110Gln Gly Thr Thr Val Thr Val
Ser Ser 115 120106112PRTArtificial SequenceCD19 VL 106Asp Ile Val
Met Thr Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly1 5 10 15Gln Pro
Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Glu Thr Ser 20 25 30Thr
Gly Asn Thr Tyr Leu Asn Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40
45Pro Gln Leu Leu Ile Tyr Arg Val Ser Lys Arg Phe Ser Gly Val Pro
50 55 60Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys
Ile65 70 75 80Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys
Leu Gln Leu 85 90 95Asp Ser Tyr Pro Asn Thr Phe Gly Gln Gly Thr Lys
Leu Glu Ile Lys 100 105 1101075PRTArtificial SequenceCD19 HCDR1
107Asp Tyr Ile Met His1 510817PRTArtificial SequenceCD19 HCDR2
108Tyr Ile Asn Pro Tyr Asn Asp Gly Ser Lys Tyr Thr Glu Lys Phe Gln1
5 10 15Gly10912PRTArtificial SequenceCD19 HCDR3 109Gly Thr Tyr Tyr
Tyr Gly Ser Glu Leu Phe Asp Tyr1 5 1011016PRTArtificial
SequenceCD19 LCDR1 110Lys Ser Ser Gln Ser Leu Glu Thr Ser Thr Gly
Asn Thr Tyr Leu Asn1 5 10 151117PRTArtificial SequenceCD19 LCDR2
111Arg Val Ser Lys Arg Phe Ser1 51129PRTArtificial SequenceCD19
LCDR3 112Leu Gln Leu Thr His Glu Pro Tyr Thr1 5113121PRTArtificial
SequenceCD19 VH 113Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys
Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr
Thr Phe Thr Asp Tyr 20 25 30Ile Met His Trp Val Arg Gln Ala Pro Gly
Gln Gly Leu Glu Trp Met 35 40 45Gly Tyr Ile Asn Pro Tyr Asn Asp Gly
Ser Lys Tyr Thr Glu Lys Phe 50 55 60Gln Gly Arg Val Thr Met Thr Ser
Asp Thr Ser Ile Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Arg Leu
Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Thr Tyr
Tyr Tyr Gly Ser Glu Leu Phe Asp Tyr Trp Gly 100 105 110Gln Gly Thr
Thr Val Thr Val Ser Ser 115 120114112PRTArtificial SequenceCD19 VL
114Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly1
5 10 15Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Glu Thr
Ser 20 25 30Thr Gly Asn Thr Tyr Leu Asn Trp Tyr Leu Gln Lys Pro Gly
Gln Ser 35 40 45Pro Gln Leu Leu Ile Tyr Arg Val Ser Lys Arg Phe Ser
Gly Val Pro 50 55 60Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe
Thr Leu Lys Ile65 70 75 80Ser Arg Val Glu Ala Glu Asp Val Gly Val
Tyr Tyr Cys Leu Gln Leu 85 90 95Thr His Glu Pro Tyr Thr Phe Gly Gln
Gly Thr Lys Leu Glu Ile Lys 100 105 110115207PRTHomo sapiens 115Met
Gln Ser Gly Thr His Trp Arg Val Leu Gly Leu Cys Leu Leu Ser1 5 10
15Val Gly Val Trp Gly Gln Asp Gly Asn Glu Glu Met Gly Gly Ile Thr
20 25 30Gln Thr Pro Tyr Lys Val Ser Ile Ser Gly Thr Thr Val Ile Leu
Thr 35 40 45Cys Pro Gln Tyr Pro Gly Ser Glu Ile Leu Trp Gln His Asn
Asp Lys 50 55 60Asn Ile Gly Gly Asp Glu Asp Asp Lys Asn Ile Gly Ser
Asp Glu Asp65 70 75 80His Leu Ser Leu Lys Glu Phe Ser Glu Leu Glu
Gln Ser Gly Tyr Tyr 85 90 95Val Cys Tyr Pro Arg Gly Ser Lys Pro Glu
Asp Ala Asn Phe Tyr Leu 100 105 110Tyr Leu Arg Ala Arg Val Cys Glu
Asn Cys Met Glu Met Asp Val Met 115 120 125Ser Val Ala Thr Ile Val
Ile Val Asp Ile Cys Ile Thr Gly Gly Leu 130 135 140Leu Leu Leu Val
Tyr Tyr Trp Ser Lys Asn Arg Lys Ala Lys Ala Lys145 150 155 160Pro
Val Thr Arg Gly Ala Gly Ala Gly Gly Arg Gln Arg Gly Gln Asn 165 170
175Lys Glu Arg Pro Pro Pro Val Pro Asn Pro Asp Tyr Glu Pro Ile Arg
180 185 190Lys Gly Gln Arg Asp Leu Tyr Ser Gly Leu Asn Gln Arg Arg
Ile 195 200 205116198PRTMacaca fascicularis 116Met Gln Ser Gly Thr
Arg Trp Arg Val Leu Gly Leu Cys Leu Leu Ser1 5 10 15Ile Gly Val Trp
Gly Gln Asp Gly Asn Glu Glu Met Gly Ser Ile Thr 20 25 30Gln Thr Pro
Tyr Gln Val Ser Ile Ser Gly Thr Thr Val Ile Leu Thr 35 40 45Cys Ser
Gln His Leu Gly Ser Glu Ala
Gln Trp Gln His Asn Gly Lys 50 55 60Asn Lys Glu Asp Ser Gly Asp Arg
Leu Phe Leu Pro Glu Phe Ser Glu65 70 75 80Met Glu Gln Ser Gly Tyr
Tyr Val Cys Tyr Pro Arg Gly Ser Asn Pro 85 90 95Glu Asp Ala Ser His
His Leu Tyr Leu Lys Ala Arg Val Cys Glu Asn 100 105 110Cys Met Glu
Met Asp Val Met Ala Val Ala Thr Ile Val Ile Val Asp 115 120 125Ile
Cys Ile Thr Leu Gly Leu Leu Leu Leu Val Tyr Tyr Trp Ser Lys 130 135
140Asn Arg Lys Ala Lys Ala Lys Pro Val Thr Arg Gly Ala Gly Ala
Gly145 150 155 160Gly Arg Gln Arg Gly Gln Asn Lys Glu Arg Pro Pro
Pro Val Pro Asn 165 170 175Pro Asp Tyr Glu Pro Ile Arg Lys Gly Gln
Gln Asp Leu Tyr Ser Gly 180 185 190Leu Asn Gln Arg Arg Ile
195117556PRTHomo sapiens 117Met Pro Pro Pro Arg Leu Leu Phe Phe Leu
Leu Phe Leu Thr Pro Met1 5 10 15Glu Val Arg Pro Glu Glu Pro Leu Val
Val Lys Val Glu Glu Gly Asp 20 25 30Asn Ala Val Leu Gln Cys Leu Lys
Gly Thr Ser Asp Gly Pro Thr Gln 35 40 45Gln Leu Thr Trp Ser Arg Glu
Ser Pro Leu Lys Pro Phe Leu Lys Leu 50 55 60Ser Leu Gly Leu Pro Gly
Leu Gly Ile His Met Arg Pro Leu Ala Ile65 70 75 80Trp Leu Phe Ile
Phe Asn Val Ser Gln Gln Met Gly Gly Phe Tyr Leu 85 90 95Cys Gln Pro
Gly Pro Pro Ser Glu Lys Ala Trp Gln Pro Gly Trp Thr 100 105 110Val
Asn Val Glu Gly Ser Gly Glu Leu Phe Arg Trp Asn Val Ser Asp 115 120
125Leu Gly Gly Leu Gly Cys Gly Leu Lys Asn Arg Ser Ser Glu Gly Pro
130 135 140Ser Ser Pro Ser Gly Lys Leu Met Ser Pro Lys Leu Tyr Val
Trp Ala145 150 155 160Lys Asp Arg Pro Glu Ile Trp Glu Gly Glu Pro
Pro Cys Leu Pro Pro 165 170 175Arg Asp Ser Leu Asn Gln Ser Leu Ser
Gln Asp Leu Thr Met Ala Pro 180 185 190Gly Ser Thr Leu Trp Leu Ser
Cys Gly Val Pro Pro Asp Ser Val Ser 195 200 205Arg Gly Pro Leu Ser
Trp Thr His Val His Pro Lys Gly Pro Lys Ser 210 215 220Leu Leu Ser
Leu Glu Leu Lys Asp Asp Arg Pro Ala Arg Asp Met Trp225 230 235
240Val Met Glu Thr Gly Leu Leu Leu Pro Arg Ala Thr Ala Gln Asp Ala
245 250 255Gly Lys Tyr Tyr Cys His Arg Gly Asn Leu Thr Met Ser Phe
His Leu 260 265 270Glu Ile Thr Ala Arg Pro Val Leu Trp His Trp Leu
Leu Arg Thr Gly 275 280 285Gly Trp Lys Val Ser Ala Val Thr Leu Ala
Tyr Leu Ile Phe Cys Leu 290 295 300Cys Ser Leu Val Gly Ile Leu His
Leu Gln Arg Ala Leu Val Leu Arg305 310 315 320Arg Lys Arg Lys Arg
Met Thr Asp Pro Thr Arg Arg Phe Phe Lys Val 325 330 335Thr Pro Pro
Pro Gly Ser Gly Pro Gln Asn Gln Tyr Gly Asn Val Leu 340 345 350Ser
Leu Pro Thr Pro Thr Ser Gly Leu Gly Arg Ala Gln Arg Trp Ala 355 360
365Ala Gly Leu Gly Gly Thr Ala Pro Ser Tyr Gly Asn Pro Ser Ser Asp
370 375 380Val Gln Ala Asp Gly Ala Leu Gly Ser Arg Ser Pro Pro Gly
Val Gly385 390 395 400Pro Glu Glu Glu Glu Gly Glu Gly Tyr Glu Glu
Pro Asp Ser Glu Glu 405 410 415Asp Ser Glu Phe Tyr Glu Asn Asp Ser
Asn Leu Gly Gln Asp Gln Leu 420 425 430Ser Gln Asp Gly Ser Gly Tyr
Glu Asn Pro Glu Asp Glu Pro Leu Gly 435 440 445Pro Glu Asp Glu Asp
Ser Phe Ser Asn Ala Glu Ser Tyr Glu Asn Glu 450 455 460Asp Glu Glu
Leu Thr Gln Pro Val Ala Arg Thr Met Asp Phe Leu Ser465 470 475
480Pro His Gly Ser Ala Trp Asp Pro Ser Arg Glu Ala Thr Ser Leu Gly
485 490 495Ser Gln Ser Tyr Glu Asp Met Arg Gly Ile Leu Tyr Ala Ala
Pro Gln 500 505 510Leu Arg Ser Ile Arg Gly Gln Pro Gly Pro Asn His
Glu Glu Asp Ala 515 520 525Asp Ser Tyr Glu Asn Met Asp Asn Pro Asp
Gly Pro Asp Pro Ala Trp 530 535 540Gly Gly Gly Gly Arg Met Gly Thr
Trp Ser Thr Arg545 550 55511810PRTArtificial Sequencelinker 118Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser1 5 1011911PRTArtificial
Sequencelinker 119Asp Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser1 5
101205PRTArtificial SequenceCD3 HVR-H1 120Asn Tyr Tyr Ile His1
512117PRTArtificial SequenceCD3 HVR-H2 121Trp Ile Tyr Pro Gly Asp
Gly Asn Thr Lys Tyr Asn Glu Lys Phe Lys1 5 10
15Gly12210PRTArtificial SequenceCD3 HVR-H3 122Asp Ser Tyr Ser Asn
Tyr Tyr Phe Asp Tyr1 5 1012317PRTArtificial SequenceCD3 HVR-L1
123Lys Ser Ser Gln Ser Leu Leu Asn Ser Arg Thr Arg Lys Asn Tyr Leu1
5 10 15Ala1247PRTArtificial SequenceCD3 HVR-L2 124Trp Ala Ser Thr
Arg Glu Ser1 51258PRTArtificial SequenceCD3 HVR-L3 125Thr Gln Ser
Phe Ile Leu Arg Thr1 5126119PRTArtificial SequenceCD3 VH 126Glu Val
Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser
Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25
30Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45Gly Trp Ile Tyr Pro Gly Asp Gly Asn Thr Lys Tyr Asn Glu Lys
Phe 50 55 60Lys Gly Arg Ala Thr Leu Thr Ala Asp Thr Ser Thr Ser Thr
Ala Tyr65 70 75 80Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95Ala Arg Asp Ser Tyr Ser Asn Tyr Tyr Phe Asp
Tyr Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val Ser Ser
115127112PRTArtificial SequenceCD3 VL 127Asp Ile Val Met Thr Gln
Ser Pro Asp Ser Leu Ala Val Ser Leu Gly1 5 10 15Glu Arg Ala Thr Ile
Asn Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30Arg Thr Arg Lys
Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45Pro Pro Lys
Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60Pro Asp
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr65 70 75
80Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Thr Gln
85 90 95Ser Phe Ile Leu Arg Thr Phe Gly Gln Gly Thr Lys Val Glu Ile
Lys 100 105 11012810PRTArtificial SequenceCD20 HVR-H1 128Gly Tyr
Thr Phe Thr Ser Tyr Asn Met His1 5 1012917PRTArtificial
SequenceCD20 HVR-H2 129Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr
Asn Gln Lys Phe Lys1 5 10 15Gly13013PRTArtificial SequenceCD20
HVR-H3 130Val Val Tyr Tyr Ser Asn Ser Tyr Trp Tyr Phe Asp Val1 5
1013110PRTArtificial SequenceCD20 HVR-L1 131Arg Ala Ser Ser Ser Val
Ser Tyr Met His1 5 101327PRTArtificial SequenceCD20 HVR-L2 132Ala
Pro Ser Asn Leu Ala Ser1 51339PRTArtificial SequenceCD20 HVR-L3
133Gln Gln Trp Ser Phe Asn Pro Pro Thr1 5134122PRTArtificial
SequenceCD20 VH 134Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val
Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr
Thr Phe Thr Ser Tyr 20 25 30Asn Met His Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Val 35 40 45Gly Ala Ile Tyr Pro Gly Asn Gly Asp
Thr Ser Tyr Asn Gln Lys Phe 50 55 60Lys Gly Arg Phe Thr Ile Ser Val
Asp Lys Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu
Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Val Val Tyr
Tyr Ser Asn Ser Tyr Trp Tyr Phe Asp Val Trp 100 105 110Gly Gln Gly
Thr Leu Val Thr Val Ser Ser 115 120135107PRTArtificial SequenceCD20
VL 135Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val
Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Ser
Tyr Met 20 25 30His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Pro
Leu Ile Tyr 35 40 45Ala Pro Ser Asn Leu Ala Ser Gly Val Pro Ser Arg
Phe Ser Gly Ser 50 55 60Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro Glu65 70 75 80Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
Trp Ser Phe Asn Pro Pro Thr 85 90 95Phe Gly Gln Gly Thr Lys Val Glu
Ile Lys Arg 100 1051365PRTMus musculus 136Asp Thr Tyr Met His1
513717PRTMus musculus 137Arg Ile Asp Pro Ala Asn Gly Asn Ser Lys
Tyr Val Pro Lys Phe Gln1 5 10 15Gly13812PRTMus musculus 138Phe Gly
Tyr Tyr Val Ser Asp Tyr Ala Met Ala Tyr1 5 1013915PRTMus musculus
139Arg Ala Gly Glu Ser Val Asp Ile Phe Gly Val Gly Phe Leu His1 5
10 151407PRTMus musculus 140Arg Ala Ser Asn Arg Ala Thr1
51419PRTMus musculus 141Gln Gln Thr Asn Glu Asp Pro Tyr Thr1
5142121PRTArtificial SequenceCEA VH 142Gln Val Gln Leu Val Gln Ser
Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys
Lys Ala Ser Gly Phe Asn Ile Lys Asp Thr 20 25 30Tyr Met His Trp Val
Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Arg Ile Asp
Pro Ala Asn Gly Asn Ser Lys Tyr Val Pro Lys Phe 50 55 60Gln Gly Arg
Val Thr Ile Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr65 70 75 80Met
Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95Ala Pro Phe Gly Tyr Tyr Val Ser Asp Tyr Ala Met Ala Tyr Trp Gly
100 105 110Gln Gly Thr Leu Val Thr Val Ser Ser 115
120143111PRTArtificial SequenceCEA VL 143Glu Ile Val Leu Thr Gln
Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly1 5 10 15Glu Arg Ala Thr Leu
Ser Cys Arg Ala Gly Glu Ser Val Asp Ile Phe 20 25 30Gly Val Gly Phe
Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro 35 40 45Arg Leu Leu
Ile Tyr Arg Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala 50 55 60Arg Phe
Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser65 70 75
80Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Thr Asn
85 90 95Glu Asp Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105 110144232PRTArtificial SequenceCD3 VH-CL 144Glu Val Gln Leu
Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr 20 25 30Ala Met
Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser
Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55
60Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr65
70 75 80Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr 85 90 95Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser
Trp Phe 100 105 110Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
Ser Ala Ser Val 115 120 125Ala Ala Pro Ser Val Phe Ile Phe Pro Pro
Ser Asp Glu Gln Leu Lys 130 135 140Ser Gly Thr Ala Ser Val Val Cys
Leu Leu Asn Asn Phe Tyr Pro Arg145 150 155 160Glu Ala Lys Val Gln
Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn 165 170 175Ser Gln Glu
Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser 180 185 190Leu
Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys 195 200
205Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr
210 215 220Lys Ser Phe Asn Arg Gly Glu Cys225
230145449PRTArtificial SequenceCEA VH-CH1(EE)-Fc (hole, P329G LALA)
145Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1
5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp
Thr 20 25 30Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu
Trp Met 35 40 45Gly Arg Ile Asp Pro Ala Asn Gly Asn Ser Lys Tyr Val
Pro Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr
Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Pro Phe Gly Tyr Tyr Val Ser Asp
Tyr Ala Met Ala Tyr Trp Gly 100 105 110Gln Gly Thr Leu Val Thr Val
Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125Val Phe Pro Leu Ala
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140Ala Leu Gly
Cys Leu Val Glu Asp Tyr Phe Pro Glu Pro Val Thr Val145 150 155
160Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
165 170 175Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
Thr Val 180 185 190Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys
Asn Val Asn His 195 200 205Lys Pro Ser Asn Thr Lys Val Asp Glu Lys
Val Glu Pro Lys Ser Cys 210 215 220Asp Lys Thr His Thr Cys Pro Pro
Cys Pro Ala Pro Glu Ala Ala Gly225 230 235 240Gly Pro Ser Val Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255Ile Ser Arg
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 260 265 270Glu
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280
285His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
290 295 300Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly305 310 315 320Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
Leu Gly Ala Pro Ile 325 330 335Glu Lys Thr Ile Ser Lys Ala Lys Gly
Gln Pro Arg Glu Pro Gln Val 340 345 350Cys Thr Leu Pro Pro Ser Arg
Asp Glu Leu Thr Lys Asn Gln Val Ser 355 360 365Leu Ser Cys Ala Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro385 390 395
400Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val
405 410 415Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
Val Met 420 425 430His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
Leu Ser Leu Ser 435 440 445Pro146674PRTArtificial SequenceCEA
VH-CH1(EE)-CD3 VL-CH1-Fc (knob, P329G LALA) 146Gln Val Gln Leu Val
Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val
Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Thr 20 25 30Tyr Met His
Trp Val Arg
Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Arg Ile Asp Pro
Ala Asn Gly Asn Ser Lys Tyr Val Pro Lys Phe 50 55 60Gln Gly Arg Val
Thr Ile Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu
Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala
Pro Phe Gly Tyr Tyr Val Ser Asp Tyr Ala Met Ala Tyr Trp Gly 100 105
110Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser
115 120 125Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly
Thr Ala 130 135 140Ala Leu Gly Cys Leu Val Glu Asp Tyr Phe Pro Glu
Pro Val Thr Val145 150 155 160Ser Trp Asn Ser Gly Ala Leu Thr Ser
Gly Val His Thr Phe Pro Ala 165 170 175Val Leu Gln Ser Ser Gly Leu
Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190Pro Ser Ser Ser Leu
Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205Lys Pro Ser
Asn Thr Lys Val Asp Glu Lys Val Glu Pro Lys Ser Cys 210 215 220Asp
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Ala Val Val Thr225 230
235 240Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly Thr Val Thr Leu
Thr 245 250 255Cys Gly Ser Ser Thr Gly Ala Val Thr Thr Ser Asn Tyr
Ala Asn Trp 260 265 270Val Gln Glu Lys Pro Gly Gln Ala Phe Arg Gly
Leu Ile Gly Gly Thr 275 280 285Asn Lys Arg Ala Pro Gly Thr Pro Ala
Arg Phe Ser Gly Ser Leu Leu 290 295 300Gly Gly Lys Ala Ala Leu Thr
Leu Ser Gly Ala Gln Pro Glu Asp Glu305 310 315 320Ala Glu Tyr Tyr
Cys Ala Leu Trp Tyr Ser Asn Leu Trp Val Phe Gly 325 330 335Gly Gly
Thr Lys Leu Thr Val Leu Ser Ser Ala Ser Thr Lys Gly Pro 340 345
350Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr
355 360 365Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro
Val Thr 370 375 380Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val
His Thr Phe Pro385 390 395 400Ala Val Leu Gln Ser Ser Gly Leu Tyr
Ser Leu Ser Ser Val Val Thr 405 410 415Val Pro Ser Ser Ser Leu Gly
Thr Gln Thr Tyr Ile Cys Asn Val Asn 420 425 430His Lys Pro Ser Asn
Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser 435 440 445Cys Asp Lys
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala 450 455 460Gly
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu465 470
475 480Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
Ser 485 490 495His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
Gly Val Glu 500 505 510Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln Tyr Asn Ser Thr 515 520 525Tyr Arg Val Val Ser Val Leu Thr Val
Leu His Gln Asp Trp Leu Asn 530 535 540Gly Lys Glu Tyr Lys Cys Lys
Val Ser Asn Lys Ala Leu Gly Ala Pro545 550 555 560Ile Glu Lys Thr
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 565 570 575Val Tyr
Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val 580 585
590Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
595 600 605Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
Thr Pro 610 615 620Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
Ser Lys Leu Thr625 630 635 640Val Asp Lys Ser Arg Trp Gln Gln Gly
Asn Val Phe Ser Cys Ser Val 645 650 655Met His Glu Ala Leu His Asn
His Tyr Thr Gln Lys Ser Leu Ser Leu 660 665 670Ser
Pro147218PRTArtificial SequenceCEA VL-CL(RK) 147Glu Ile Val Leu Thr
Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly1 5 10 15Glu Arg Ala Thr
Leu Ser Cys Arg Ala Gly Glu Ser Val Asp Ile Phe 20 25 30Gly Val Gly
Phe Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro 35 40 45Arg Leu
Leu Ile Tyr Arg Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala 50 55 60Arg
Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser65 70 75
80Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Thr Asn
85 90 95Glu Asp Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
Arg 100 105 110Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser
Asp Arg Lys 115 120 125Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu
Leu Asn Asn Phe Tyr 130 135 140Pro Arg Glu Ala Lys Val Gln Trp Lys
Val Asp Asn Ala Leu Gln Ser145 150 155 160Gly Asn Ser Gln Glu Ser
Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 165 170 175Tyr Ser Leu Ser
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 180 185 190His Lys
Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 195 200
205Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210
215148449PRTArtificial SequenceCD19 VH-CH1(EE)-Fc(hole, P329G LALA)
148Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1
5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp
Tyr 20 25 30Ile Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu
Trp Met 35 40 45Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Ser Lys Tyr Thr
Glu Lys Phe 50 55 60Gln Gly Arg Val Thr Met Thr Ser Asp Thr Ser Ile
Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Arg Leu Arg Ser Asp Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Thr Tyr Tyr Tyr Gly Pro
Gln Leu Phe Asp Tyr Trp Gly 100 105 110Gln Gly Thr Thr Val Thr Val
Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125Val Phe Pro Leu Ala
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140Ala Leu Gly
Cys Leu Val Glu Asp Tyr Phe Pro Glu Pro Val Thr Val145 150 155
160Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
165 170 175Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
Thr Val 180 185 190Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys
Asn Val Asn His 195 200 205Lys Pro Ser Asn Thr Lys Val Asp Glu Lys
Val Glu Pro Lys Ser Cys 210 215 220Asp Lys Thr His Thr Cys Pro Pro
Cys Pro Ala Pro Glu Ala Ala Gly225 230 235 240Gly Pro Ser Val Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255Ile Ser Arg
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 260 265 270Glu
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280
285His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
290 295 300Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly305 310 315 320Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
Leu Gly Ala Pro Ile 325 330 335Glu Lys Thr Ile Ser Lys Ala Lys Gly
Gln Pro Arg Glu Pro Gln Val 340 345 350Cys Thr Leu Pro Pro Ser Arg
Asp Glu Leu Thr Lys Asn Gln Val Ser 355 360 365Leu Ser Cys Ala Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro385 390 395
400Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val
405 410 415Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
Val Met 420 425 430His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
Leu Ser Leu Ser 435 440 445Pro149674PRTArtificial SequenceCD19
VH-CH1(EE)-CD3 VL-CH1-Fc(knob, P329G LALA) 149Gln Val Gln Leu Val
Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val
Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30Ile Met His
Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Tyr
Ile Asn Pro Tyr Asn Asp Gly Ser Lys Tyr Thr Glu Lys Phe 50 55 60Gln
Gly Arg Val Thr Met Thr Ser Asp Thr Ser Ile Ser Thr Ala Tyr65 70 75
80Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Gly Thr Tyr Tyr Tyr Gly Pro Gln Leu Phe Asp Tyr Trp
Gly 100 105 110Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys
Gly Pro Ser 115 120 125Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr
Ser Gly Gly Thr Ala 130 135 140Ala Leu Gly Cys Leu Val Glu Asp Tyr
Phe Pro Glu Pro Val Thr Val145 150 155 160Ser Trp Asn Ser Gly Ala
Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175Val Leu Gln Ser
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190Pro Ser
Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200
205Lys Pro Ser Asn Thr Lys Val Asp Glu Lys Val Glu Pro Lys Ser Cys
210 215 220Asp Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Ala Val
Val Thr225 230 235 240Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly
Thr Val Thr Leu Thr 245 250 255Cys Gly Ser Ser Thr Gly Ala Val Thr
Thr Ser Asn Tyr Ala Asn Trp 260 265 270Val Gln Glu Lys Pro Gly Gln
Ala Phe Arg Gly Leu Ile Gly Gly Thr 275 280 285Asn Lys Arg Ala Pro
Gly Thr Pro Ala Arg Phe Ser Gly Ser Leu Leu 290 295 300Gly Gly Lys
Ala Ala Leu Thr Leu Ser Gly Ala Gln Pro Glu Asp Glu305 310 315
320Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn Leu Trp Val Phe Gly
325 330 335Gly Gly Thr Lys Leu Thr Val Leu Ser Ser Ala Ser Thr Lys
Gly Pro 340 345 350Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr
Ser Gly Gly Thr 355 360 365Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
Phe Pro Glu Pro Val Thr 370 375 380Val Ser Trp Asn Ser Gly Ala Leu
Thr Ser Gly Val His Thr Phe Pro385 390 395 400Ala Val Leu Gln Ser
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 405 410 415Val Pro Ser
Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 420 425 430His
Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser 435 440
445Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala
450 455 460Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
Thr Leu465 470 475 480Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
Val Val Asp Val Ser 485 490 495His Glu Asp Pro Glu Val Lys Phe Asn
Trp Tyr Val Asp Gly Val Glu 500 505 510Val His Asn Ala Lys Thr Lys
Pro Arg Glu Glu Gln Tyr Asn Ser Thr 515 520 525Tyr Arg Val Val Ser
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 530 535 540Gly Lys Glu
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro545 550 555
560Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
565 570 575Val Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn
Gln Val 580 585 590Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser
Asp Ile Ala Val 595 600 605Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro 610 615 620Pro Val Leu Asp Ser Asp Gly Ser
Phe Phe Leu Tyr Ser Lys Leu Thr625 630 635 640Val Asp Lys Ser Arg
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 645 650 655Met His Glu
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 660 665 670Ser
Pro150219PRTArtificial SequenceCD19 VL-CL(RK) 150Asp Ile Val Met
Thr Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly1 5 10 15Gln Pro Ala
Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Glu Thr Ser 20 25 30Thr Gly
Thr Thr Tyr Leu Asn Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45Pro
Gln Leu Leu Ile Tyr Arg Val Ser Lys Arg Phe Ser Gly Val Pro 50 55
60Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile65
70 75 80Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Leu Gln
Leu 85 90 95Leu Glu Asp Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu
Ile Lys 100 105 110Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro
Pro Ser Asp Arg 115 120 125Lys Leu Lys Ser Gly Thr Ala Ser Val Val
Cys Leu Leu Asn Asn Phe 130 135 140Tyr Pro Arg Glu Ala Lys Val Gln
Trp Lys Val Asp Asn Ala Leu Gln145 150 155 160Ser Gly Asn Ser Gln
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 165 170 175Thr Tyr Ser
Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 180 185 190Lys
His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 195 200
205Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215
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