U.S. patent application number 10/372481 was filed with the patent office on 2003-10-30 for reagents and treatment methods for autoimmune diseases.
Invention is credited to Tedder, Thomas F..
Application Number | 20030202975 10/372481 |
Document ID | / |
Family ID | 27767572 |
Filed Date | 2003-10-30 |
United States Patent
Application |
20030202975 |
Kind Code |
A1 |
Tedder, Thomas F. |
October 30, 2003 |
Reagents and treatment methods for autoimmune diseases
Abstract
The invention concerns treatment methods using anti-CD22
monoclonal antibodies with unique physiologic properties. In
particular, the invention concerns methods for the treatment of
B-cell malignancies and autoimmune diseases by administering an
effective amount of a blocking anti-CD22 monoclonal antibody
specifically binding to the first two Ig-like domains, or to an
epitope within the first two Ig-like domains of native human CD22
(hCD22).
Inventors: |
Tedder, Thomas F.; (Durham,
NC) |
Correspondence
Address: |
MYERS BIGEL SIBLEY & SAJOVEC
PO BOX 37428
RALEIGH
NC
27627
US
|
Family ID: |
27767572 |
Appl. No.: |
10/372481 |
Filed: |
February 21, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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60359419 |
Feb 21, 2002 |
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60420472 |
Oct 21, 2002 |
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Current U.S.
Class: |
424/144.1 ;
435/7.1 |
Current CPC
Class: |
A61P 13/12 20180101;
A61K 2039/505 20130101; A61P 1/04 20180101; A61P 19/00 20180101;
A61P 27/02 20180101; C07K 2317/56 20130101; A61P 13/02 20180101;
A61P 5/14 20180101; A61P 21/00 20180101; A61P 7/06 20180101; A61P
35/02 20180101; A61P 7/04 20180101; A61P 17/00 20180101; A61P 35/00
20180101; A61P 37/00 20180101; A61P 37/04 20180101; A61P 17/02
20180101; C07K 16/2803 20130101; A61P 37/08 20180101; A61P 7/00
20180101; A61P 25/00 20180101; A61P 37/02 20180101; A61P 21/04
20180101; A61P 7/02 20180101; A61P 1/00 20180101; A61P 5/16
20180101; A61K 39/39533 20130101; A61P 9/10 20180101; A61P 19/04
20180101; A61P 5/00 20180101; A61P 29/00 20180101; A61P 3/10
20180101; A61P 17/06 20180101; C07K 2317/76 20130101; A61P 9/00
20180101; A61P 19/02 20180101; A61P 37/06 20180101; C07K 2317/73
20130101; A61P 9/08 20180101; A61K 39/39533 20130101; A61K 2300/00
20130101 |
Class at
Publication: |
424/144.1 ;
435/7.1 |
International
Class: |
G01N 033/53; A61K
039/395 |
Goverment Interests
[0002] The present invention was made with the support of Grant No.
CA 81776 from the National Institutes of Health. The United States
government has certain rights in this invention.
Claims
What is claimed is:
1. A method for treating a human patient diagnosed with an
autoimmune disease, comprising (1) administering to said human
patient an effective amount of a blocking anti-CD22 monoclonal
antibody wherein said antibody comprises a heavy chain comprising a
V.sub.H sequence having at least about 95% sequence identity with
the sequence of amino acids 1 to 100 of SEQ ID NO: 9 (HB22-5
V.sub.H sequence); or amino acids 1 to 97 of SEQ ID NO: 11 (HB22-7
V.sub.H sequence); or amino acids I to 100 of SEQ ID NO: 13
(HB22-13 V.sub.H sequence); or amino acids 1 to 100 of SEQ ID NO:
15 (HB22-23 V.sub.H sequence); or amino acids 1 to 98 of SEQ ID NO:
17 (HB22-33 V.sub.H sequence); or amino acids 1 to 100 of SEQ ID
NO: 19 (HB22-196 V.sub.H sequence); and (2) monitoring the response
of said autoimmune disease to said treatment.
2. The method of claim 1 wherein said antibody comprises a heavy
chain comprising a V.sub.H sequence having at least about 95%
sequence identity with the sequence of amino acids 1 to 97 of SEQ
ID NO: 11 (HB22-7 V.sub.H sequence); or amino acids 1 to 100 of SEQ
ID NO: 15 (HB22-23 V.sub.H sequence); or amino acids 1 to 98 of SEQ
ID NO: 17 (HB22-33 V.sub.H sequence).
3. The method of claim 2 wherein said antibody comprises a V.sub.H
sequence selected from the group consisting of amino acids 1 to 97
of SEQ ID NO: 11 (HB22-7 V.sub.H sequence); amino acids 1 to 100 of
SEQ ID NO: 15 (HB22-23 V.sub.H sequence); and amino acids 1 to 98
of SEQ ID NO: 17 (HB22-33 V.sub.H sequence).
4. A method for treating a human patient diagnosed with an
autoimmune disease, comprising (1) administering to said human
patient an effective amount of a blocking anti-CD22 monoclonal
antibody wherein said antibody comprises a light chain comprising a
V.sub..kappa. sequence having at least about 95% sequence identity
with the amino acid sequence of SEQ ID NO: 21 (HB22-5 V.sub..kappa.
sequence); or SEQ ID NO: 23 (HB22-7 V.sub..kappa. sequence); or SEQ
ID NO: 25 (HB22-13 V.sub..kappa. sequence); or SEQ ID NO: 27
(HB22-23 V.sub..kappa. sequence); or SEQ ID NO: 29 (HB22-33
V.sub..kappa. sequence); or SEQ ID NO: 31 (HB22-196 V.sub..kappa.
sequence); and (2) monitoring the response of said autoimmune
disease to said treatment.
5. The method of claim 4 wherein said antibody comprises a light
chain comprising a V.sub..kappa. sequence having at least about 95%
sequence identity with the amino acid sequence of SEQ ID NO: 23
(HB22-7 V.sub..kappa. sequence); or SEQ ID NO: 27 (HB22-23
V.sub..kappa. sequence); or SEQ ID NO: 29 (HB22-33 V.sub..kappa.
sequence).
6. The method of claim 5 wherein said antibody comprises a
V.sub..kappa. sequence selected from the group consisting of the
amino acid sequence of SEQ ID NO: 23 (HB22-7 V.sub..kappa.
sequence); SEQ ID NO: 27 (HB22-23 V.sub..kappa. sequence); and SEQ
ID NO: 29 (HB22-33 V.sub..kappa. sequence).
7. The method of claim 1 wherein said antibody comprises V.sub.H
and V.sub..kappa. sequences selected from the group consisting of
amino acids 1 to 97 of SEQ ID NO: 11 (HB22-7 V.sub.H sequence) and
the amino acid sequence of SEQ ID NO: 23 (HB22-7 V.sub..kappa.
sequence); amino acids 1 to 100 of SEQ ID NO: 15 (HB22-23 V.sub.H
sequence) and the amino acid sequence of SEQ ID NO: 27 (HB22-23
V.sub..kappa. sequence); and amino acids 1 to 98 of SEQ ID NO: 17
(HB22-33 V.sub.H sequence) and the amino acid sequence of SEQ ID
NO: 29 (HB22-33 V.sub..kappa. sequence).
8. The method of claim 1 wherein said treatment is unaccompanied by
any other treatment for the autoimmune disease.
9. The method of claim 1 wherein said antibody is a fragment of a
complete antibody.
10. The method of claim 9 wherein said antibody is selected from
the group consisting of Fab, Fab', F(ab').sub.2, and Fv fragments,
diabodies, linear antibodies, single-chain antibody molecules, and
multispecific antibodies formed from antibody fragments.
11. The method of claim 1 wherein said antibody comprises
antigen-specificity and is effective to bind to the first two
Ig-like domains of a CD22 molecule, or to bind to an epitope within
the first two Ig-like domains of native human CD22 (hCD22) of SEQ
ID NO: 1, and further has an additional antigen-specificity.
12. The method of claim 10 wherein said antibody is a bispecific
antibody.
13. The method of claim 12 wherein said antibody additionally binds
to another epitope of CD22.
14. The method of claim 1 wherein said antibody is chimeric.
15. The method of claim 1 wherein said antibody is humanized.
16. The method of claim 1 wherein said antibody is human.
17. The method of claim 1 wherein said antibody is administered
intravenously.
18. The method of claim 17 wherein said antibody is administered by
weekly intravenous infusions.
19. The method of claim 1, wherein said human patient is further
administered an anti-CD20 antibody.
20. The method of claim 7 wherein said antibody is chimeric.
21. The method of claim 7 wherein said antibody is humanized.
22. The method of claim 7 wherein said antibody is human.
23. The method of claim 4 wherein said treatment is unaccompanied
by any other treatment for the autoimmune disease.
24. The method of claim 4 wherein said antibody is a fragment of a
complete antibody.
25. The method of claim 24 wherein said antibody is selected from
the group consisting of Fab, Fab', F(ab').sub.2, and Fv fragments,
diabodies, linear antibodies, single-chain antibody molecules, and
multispecific antibodies formed from antibody fragments.
26. The method of claim 4 wherein said antibody comprises
antigen-specificity and is effective to bind to the first two
Ig-like domains of a CD22 molecule, or to bind to an epitope within
the first two Ig-like domains of native human CD22 (hCD22) of SEQ
ID NO: 1, and further has an additional antigen-specificity.
27. The method of claim 25 wherein said antibody is a bispecific
antibody.
28. The method of claim 27 wherein said antibody additionally binds
to another epitope of CD22.
29. The method of claim 4 wherein said antibody is chimeric.
30. The method of claim 4 wherein said antibody is humanized.
31. The method of claim 4 wherein said antibody is human.
32. The method of claim 4 wherein said antibody is administered
intravenously.
33. The method of claim 32 wherein said antibody is administered by
weekly intravenous infusions.
34. The method of claim 4, wherein said human patient is further
administered an anti-CD20 antibody.
35. An isolated nucleic acid molecule comprising nucleic acid
encoding an antibody heavy chain variable region comprising a
V.sub.H sequence having at least about 95% sequence identity with
the sequence of amino acids 1 to 100 of SEQ ID NO: 9 (HB22-5
V.sub.H sequence); or amino acids 1 to 97 of SEQ ID NO: 11 (HB22-7
V.sub.H sequence); or amino acids 1 to 100 of SEQ ID NO: 13
(HB22-13 V.sub.H sequence); or amino acids 1 to 100 of SEQ ID NO:
15 (HB22-23 V.sub.H sequence); or amino acids 1 to 98 of SEQ ID NO:
17 (HB22-33 V.sub.H sequence); or amino acids 1 to 100 of SEQ ID
NO: 19 (HB22-196 V.sub.H sequence).
36. An isolated nucleic acid molecule comprising nucleic acid
encoding an antibody light chain variable region comprising a
V.sub..kappa. sequence having at least about 95% sequence identity
with the amino acid sequence of SEQ ID NO: 21 (HB22-5 V.sub..kappa.
sequence); or SEQ ID NO: 23 (HB22-7 V.sub..kappa. sequence); or SEQ
ID NO: 25 (HB22-13 V.sub..kappa. sequence); or SEQ ID NO: 27
(HB22-23 V.sub..kappa. sequence); or SEQ ID NO: 29 (HB22-33
V.sub..kappa. sequence); or SEQ ID NO: 31 (HB22-196 V.sub..kappa.
sequence).
37. An isolated nucleic acid molecule comprising nucleic acid
encoding a V.sub.H sequence selected from the group consisting of
amino acids 1 to 97 of SEQ ID NO: 11 (HB22-7 V.sub.H sequence);
amino acids 1 to 100 of SEQ ID NO: 15 (HB22-23 V.sub.H sequence);
and amino acids 1 to 98 of SEQ ID NO: 17 (HB22-33 V.sub.H
sequence).
38. An isolated nucleic acid molecule comprising nucleic acid
encoding a V.sub..kappa. sequence selected from the group
consisting of the amino acid sequence of SEQ ID NO: 23 (HB22-7
V.sub..kappa. sequence); SEQ ID NO: 27 (HB22-23 V.sub..kappa.
sequence); and SEQ ID NO: 29 (HB22-33 V.sub..kappa. sequence).
Description
[0001] This application claims priority from U.S. Provisional
Application Serial No. 60/359,419, filed Feb. 21, 2002 and U.S.
Provisional Application Serial No. 60/420,472, filed Oct. 21, 2002,
both of which applications are hereby incorporated by reference in
their entireties.
BACKGROUND OF THE INVENTION
[0003] 1. Field of the Invention
[0004] The present invention concerns the therapeutic use of
certain anti-CD22 monoclonal antibodies with unique physiologic
properties. More specifically, the invention concerns methods of
treating B-cell malignancies, such as lymphomas and leukemias, and
autoimmune diseases with blocking anti-CD22 antibodies having
unique pro-apoptotic properties.
[0005] 2. Description of the Related Art
[0006] CD22 is a membrane glycophosphoprotein found on nearly all B
lymphocytes and most B-cell lymphomas. Cross-linking CD22 triggers
CD22 tyrosine phosphorylation and assembles a complex of effector
proteins that activate the stress-activated protein kinase (SAPK)
pathway. CD22 cross-linking provides a potent costimulatory signal
in primary B-cells and pro-apoptotic signal in neoplastic B-cells.
Structurally, CD22 is a member of the "sialoadhesin" subclass of
the immunoglobulin (Ig) gene superfamily, having seven
extracellular Ig domains with a single amino-terminal V-set Ig
domain and six C-2 set Ig domains. Wilson et al., J. Exp. Med.
173:137-146 (1991); Engel et al., J. Exp. Med. 181:1581-1586
(1995); and Torres et al., J. Immunol. 149:2641-2649 (1992). It has
been shown that CD22 is a critical lymphocyte-specific signal
transduction molecule which negatively and positively regulates B
lymphocyte antigen receptor (BCR) signaling by recruiting signaling
effector molecules to physiologically pertinent sites. Tedder et
al., Annu. Rev. Immunol. 15:481-504 (1997); Sato et al., Immunology
10:287-297 (1998).
[0007] Anti-CD22 antibodies have been described, for example in
U.S. Pat. Nos. 5,484,892; 6,183,744; 6,187,287; 6,254,868, and in
Tuscano et al., Blood 94(4):1382-92 (1999). The use of monoclonal
antibodies, including anti-CD22 antibodies, in the treatment of
non-Hodgkin's lymphoma is reviewed, for example, by Renner et al.,
Leukemia 11(Suppl. 2):S55-9 (1997). A humanized anti-CD22 antibody,
LymphoCide.TM. (empatuzumab, Immunomedics, Inc.) is in Phase III
clinical trials for the treatment of indolent and aggressive forms
of non-Hodgkin's lymphomas. An yttrium-90-labeled version of this
antibody is currently in Phase I clinical trials for the same
indication.
[0008] Despite recent advances in cancer therapy, B-cell
malignancies, such as the B-cell subtype of non-Hodgkin's lymphoma,
and chronic lymphocytic leukemia, are major contributors of
cancer-related deaths. Accordingly, there is a great need for
further, improved therapeutic regimens for the treatment of B-cell
malignancies. Autoimmune diseases as a whole cause significant
morbidity and disability. Based on incidence data collected from
1965 to 1995, it has been estimated that approximately 1,186,015
persons will develop a new autoimmune disease over the next 5
years. Jacobsen et al. (Clin. Immunol. Immunopathol. 84:223 (1997))
evaluated over 130 published studies and estimated that in 1996,
8.5 million people in the United States (3.2% of the population)
had at least one of the 24 autoimmune diseases examined in these
studies. Considering the major impact of autoimmune diseases on
public health, effective and safe treatments are needed to address
the burden of these disorders. Thus, there is a need in the art for
improved reagents and methods for treating autoimmune disease.
SUMMARY OF THE INVENTION
[0009] The present invention concerns an improved clinical approach
for the treatment of B-cell malignancies and autoimmune disease in
human patients, taking advantage of the unique properties of
certain blocking anti-CD22 monoclonal antibodies.
[0010] In one aspect, the invention concerns a method for treating
a human patient diagnosed with a B-cell malignancy, comprising (1)
administering to the patient an effective amount of a blocking
anti-CD22 monoclonal antibody specifically binding to the first two
Ig-like domains or to an epitope associated with the first two
Ig-like domains of native human CD22 (hCD22) of SEQ ID NO: 1, and
(2) monitoring the response of the malignancy to the treatment.
[0011] In a further aspect, the invention concerns a method for
treating a subject (e.g., a human patient) diagnosed with an
autoimmune disease, comprising (1) administering to the subject an
effective amount of a blocking anti-CD22 monoclonal antibody and,
optionally, (2) monitoring the response of the autoimmune disease
to the treatment.
[0012] As further aspects, the present invention provides methods
of reducing B cell activity, reducing the number of B cells or B
cell subsets or even essentially eliminating B cells or particular
B cell subsets, increasing turnover of B cells and/or reducing
antibody production by B cells by administering to a subject (e.g.,
a human patient) an effective amount of a blocking anti-CD22
monoclonal antibody; and optionally, (2) monitoring the response to
the treatment. By "reducing" it is meant at least about a 25%, 35%,
50% or 75% decrease or more. By "essentially eliminating" it is
meant at least about a 90%, 95%, 985 or more decrease or more. By
"increasing turnover", it is meant at least about a 25%, 35%, 50%,
75%, 100%, 150% or more elevation in turnover rate.
[0013] In a particular embodiment, the antibody used binds to
essentially the same epitope as an antibody selected from the group
consisting of HB22-7 (HB11347), HB22-23 (HB11349), HB22-33, HB22-5,
HB22-13, and HB22-196, preferably HB22-7, HB22-23, or HB22-33, more
preferably HB22-7 or HB22-33.
[0014] In a further embodiment, the antibody blocks CD22 binding to
its ligand by at least 70%, preferably by at least 80%.
[0015] In another embodiment, the antibody comprises a heavy chain
comprising a V.sub.H sequence having at least about 95% sequence
identity with the sequence of amino acids 1 to 100 of SEQ ID NO: 9
(HB22-5 V.sub.H sequence); or amino acids 1 to 97 of SEQ ID NO: 11
(HB22-7 V.sub.H sequence); or amino acids 1 to 100 of SEQ ID NO: 13
(HB22-13 V.sub.H sequence); or amino acids 1 to 100 of SEQ ID NO:
15 (HB22-23 V.sub.H sequence); or amino acids 1 to 98 of SEQ ID NO:
17 (HB22-33 V.sub.H sequence); or amino acids 1 to 100 of SEQ ID
NO: 19 (HB22-196 V.sub.H sequence).
[0016] In yet another embodiment, the antibody comprises a heavy
chain comprising a V.sub.H sequence having at least about 95%
sequence identity with the sequence of amino acids 1 to 97 of SEQ
ID NO: 11 (HB22-7 V.sub.H sequence); or amino acids 1 to 100 of SEQ
ID NO: 15 (HB22-23 V.sub.H sequence); or amino acids 1 to 98 of SEQ
ID NO: 17 (HB22-33 V.sub.H sequence).
[0017] In a still further embodiment, the antibody comprises a
V.sub.H sequence selected from the group consisting of amino acids
1 to 97 of SEQ ID NO: 11 (HB22-7 V.sub.H sequence); amino acids 1
to 100 of SEQ ID NO: 15 (HB22-23 V.sub.H sequence); and amino acids
1 to 98 of SEQ ID NO: 17 (HB22-33 V.sub.H sequence).
[0018] In a different embodiment, the antibody comprises a light
chain comprising a V.sub..kappa. sequence having at least about 95%
sequence identity with the amino acid sequence of SEQ ID NO: 21
(HB22-5 V.sub..kappa. sequence); or SEQ ID NO: 23 (HB22-7
V.sub..kappa. sequence); or SEQ ID NO: 25 (HB22-13 V.sub..kappa.
sequence); or SEQ ID NO: 27 (HB22-23 V.sub..kappa. sequence); or
SEQ ID NO: 29 (HB22-33 V.sub..kappa. sequence); or SEQ ID NO: 31
(HB22-196 V.sub..kappa. sequence).
[0019] In a particular embodiment, the antibody comprises a light
chain comprising a V.sub..kappa. sequence having at least about 95%
sequence identity with the amino acid sequence of SEQ ID NO: 23
(HB22-7 V.sub..kappa. sequence); or SEQ ID NO: 27 (HB22-23
V.sub..kappa. sequence); or SEQ ID NO: 29 (HB22-33 V.sub..kappa.
sequence).
[0020] In a further embodiment, the antibody comprises a
V.sub..kappa. sequence selected from the group consisting of the
amino acid sequence of SEQ ID NO: 23 (HB22-7 V.sub..kappa.
sequence); SEQ ID NO: 27 (HB22-23 V.sub..kappa. sequence); and SEQ
ID NO: 29 (HB22-33 V.sub..kappa. sequence).
[0021] In a preferred embodiment, the antibody comprises V.sub.H
and V.sub..kappa. sequences selected from the group consisting of
amino acids 1 to 97 of SEQ ID NO: 11 (HB22-7 V.sub.H sequence) and
the amino acid sequence of SEQ ID NO: 23 (HB22-7 V.sub..kappa.
sequence); amino acids 1 to 100 of SEQ ID NO: 15 (HB22-23 V.sub.H
sequence) and the amino acid sequence of SEQ ID NO: 27 (HB22-23
V.sub..kappa. sequence); and amino acids 1 to 98 of SEQ ID NO: 17
(HB22-33 V.sub.H sequence) and the amino acid sequence of SEQ ID
NO: 29 (HB22-33 V.sub..kappa. sequence).
[0022] In a different aspect, the invention concerns nucleic acid
encoding any of the antibody heavy or light chain variable regions
discussed above, or any portion thereof.
[0023] As a further aspect the present invention provides
polypeptides comprising the heavy or light chain variable regions
discussed above, or a portion thereof.
[0024] The targeted condition can be any type of autoimmune disease
or B-cell malignancy, including but not limited to localized B-cell
malignancies, or any other condition in which B cells or antibodies
are implicated. Typical representatives of B-cell malignancies are
B-cell subtype of non-Hodgkin's lymphoma, Burkitt's lymphoma,
multiple myeloma, chronic lymphocytic leukemia, hairy cell
leukemia, and prolymphocytic leukemia.
[0025] The treatment methods of the present invention may be
performed without any further treatment of malignant B cells or
autoimmune disease. With respect to B-cell malignancy, the
treatment method of the present invention typically provides
improved cure rate and/or increased survival and/or superior tumor
volume reduction when compared to no treatment, combination
treatment with the same antibody and radioimmunotherapy, or with
radioimmunotherapy alone.
[0026] The antibody can be a complete antibody, or an antibody
fragment, including, for example, Fab, Fab', F(ab').sub.2, and Fv
fragments, diabodies, linear antibodies, single-chain antibody
molecules, and multispecific antibodies formed from antibody
fragments. Thus, the antibody may have an additional antigen
specificity, e.g. may be a bispecific antibody. The bispecific
antibody may, for example, additionally bind to another epitope to
CD22. In addition, the bispecific antibody may have binding
specificity for other antigens, such as, CD19, CD20, CD52, CD3,
CD28, or HLA-DR10 (Lym-1); or for Fc receptors, e.g. CD16, CD64 and
CD89.
[0027] The antibody may be chimeric, humanized, primatized, or
human.
[0028] The administration of the antibody may be performed by any
conventional route, such as intravenous (i.v.) administration by
repeated intravenous infusions.
[0029] The response to the treatment may be monitored by methods
well known for a skilled practitioner, including monitoring
shrinkage of a solid tumor, e.g. by magnetic resonance imaging
(MRI), or by measuring improvement or stabilization in clinical
indicia of autoimmune disease, as known by those skilled in the
art.
BRIEF DESCRIPTION OF THE DRAWINGS
[0030] FIG. 1 shows the amino acid sequence of human CD22 (hCD22),
where the boundaries of the Ig-like domains (domains 1-7) are
indicated
[0031] FIG. 2. Whole body autoradiography of Raji and Ramos
tumor-bearing nude mice injected with .sup.111In-2IT-BAD-antiCD22
(HB22-7). Mice were sacrificed and autoradiographed 48 hours after
injection. Upper image is Raji-tumored mouse, lower image is
Ramos-tumored mouse.
[0032] FIG. 3. The temporal assessment of tumor volume in
Raji-xenografted mice that were untreated or treated with 125 uCi
.sup.90Y-DOTA-peptide-Ly- m-1 (RIT) alone, anti-CD22 alone
(HB22-7), or three different sequences of RIT and HB22-7 (CMRIT) in
trial 081500. Tumor volume was assessed three times per week. Mouse
numbers for each treatment group are tabulated (Table 2).
[0033] FIG. 4. Summary analysis of tumor volume observed in all
independent xenograft trials. The trials were conducted as
described in FIG. 2. Mouse numbers for each trial are tabulated
(Table 2).
[0034] FIG. 5. The response and cure rate for Raji-xenografted mice
that were treated as described in FIG. 2. The tumor responses were
categorized as follows: C, cure (tumor disappeared and did not
regrow by the end of the 84-day study); CR, complete regression
(tumor disappeared for at least 7 days but later regrew); PR,
partial regression (tumor volume decreased by 50% or more for at
least 7 days, then regrew). The data represents results of all
independent trials.
[0035] FIG. 6. Overall survival was assessed for Raji xenografted
mice that were treated as described in FIG. 2. Mice were euthanized
when the tumor burden exceeded 2000 mg or at the end of the 84 day
trial. The data represents results of all independent trials.
[0036] FIGS. 7a, 7b and 7c. Hematologic toxicity was assessed by
measuring white blood cell (WBC) (FIG. 7b), red blood cell (RBC)
(FIG. 7c) and platelet counts (FIG. 7a) twice weekly in the
Raji-xenografted mice that were treated as described in FIG. 2.
When compared to RIT alone there was no difference in hematologic
toxicity in the CMRIT groups. In addition, there was no hematologic
toxicity observed in the mice treated with HB22-7 alone.
[0037] FIG. 8. Non-hematologic toxicity was assessed by measuring
body weights twice weekly in Raji xenografted mice that were
treated as described in FIG. 2. There were no significant
differences in body weights in any of the treatment groups in all
five xenograft trials.
[0038] FIG. 9. RIT clearance was assessed by measuring
radioactivity in whole body (WB) and blood daily for 5 days after
initiation of treatment with RIT. The results were reported after
adjusting for decay based on the T.sub.1/2 of .sup.90Y. There were
no significant differences in RIT clearance in any of the CMRIT
treatment groups.
[0039] FIG. 10. V.sub.H amino acid sequence analysis of anti-CD22
antibodies (Abs) that block ligand binding. Amino acid numbering
and designations of the origins of the coding sequence for each Ab
is according to the convention of Kabat et al. (Sequences of
Proteins of Immunological Interest, U.S. Government Printing
Office, Bethesda, Md., 1991), where amino acid positions 1-94, CDR1
and 2, and FR1, 2, and 3 are encoded by a V.sub.H gene. Sequences
that overlap with the 5' PCR primers are not shown. A dot indicates
a gap inserted in the sequence to maximize alignment of similar
amino acid sequences. Gaps in the sequences were introduced between
V.sub.H, D and J segments for clarity. The rank order of sequences
shown was based on relatedness to the HB22-5 sequence.
[0040] FIGS. 11-16. Nucleotide and encoded amino acid sequences for
heavy chain V.sub.H-D-J.sub.H junctional sequences for anti-CD22
Abs from hybridomas HB22-5 (SEQ ID NOS: 8 and 9), HB22-7 (SEQ ID
NOS: 10 and I 1); HB22-13 (SEQ ID NOS: 12 and 13); HB22-23 (SEQ ID
NOS: 14 and 15); HB22-33 (SEQ ID NOS: 16 and 17); and HB22-196 (SEQ
ID NOS: 18 and 19). Sequences that overlap with the 5' PCR primers
are indicated by double underlining. D region sequences are
underlined.
[0041] FIG. 17. Light chain V.kappa. amino acid sequence analysis
of anti-CD22 Abs that block ligand binding. Amino acid numbering
and designation of origins of the coding sequence for each Ab is
according to the convention of Kabat et al., supra. The amino acid
following the predicted signal sequence cleavage site is numbered
1. A dot indicates a gap inserted in the sequence to maximize
alignment of similar amino acid sequences. Gaps in the sequences
were introduced between V.kappa., J segments and .kappa. constant
region (double underlined) sequences for clarity.
[0042] FIGS. 18-23. Nucleotide and deduced amino acid sequences for
kappa light chain V-J-constant region junctional sequences for
anti-CD22 Abs from hybridomas HB22-5 (SEQ ID NOS: 20 and 21);
HB22-7 (SEQ ID NOS: 22 and 23): HB22-13 (SEQ ID NOS: 24 and 25):
HB22-23 (SEQ ID NOS: 26 and 27); HB22-33 (SEQ ID NOS: 28 and 29);
and HB22-196 (SEQ ID NOS: 30 and 31). Sequences that overlap with
the 5' PCR primers are indicated by double underlining.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0043] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. The
terminology used in the description of the invention herein is for
the purpose of describing particular embodiments only and is not
intended to be limiting of the invention.
[0044] All publications, patent applications, patents, and other
references mentioned herein are incorporated by reference in their
entirety.
A. Definitions
[0045] Unless defined otherwise, technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs.
[0046] One skilled in the art will recognize many methods and
materials similar or equivalent to those described herein, which
could be used in the practice of the present invention. Indeed, the
present invention is in no way limited to the methods and materials
described. For purposes of the present invention, the following
terms are defined below.
[0047] The term "immunoglobulin" (Ig) is used to refer to the
immunity-conferring portion of the globulin proteins of serum, and
to other glycoproteins, which may not occur in nature but have the
same functional characteristics. The term "immunoglobulin" or "Ig"
specifically includes "antibodies" (Abs). While antibodies exhibit
binding specificity to a specific antigen, immunoglobulins include
both antibodies and other antibody-like molecules that lack antigen
specificity. Native immunoglobulins are secreted by differentiated
B cells termed plasma cells, and immunoglobulins without any
antigen specificity are produced at low levels by the lymph system
and at increased levels by myelomas. As used herein, the terms
"immunoglobulin," "Ig," and grammatical variants thereof are used
to include antibodies (as hereinabove defined), and Ig molecules
without antigen specificity.
[0048] Native immunoglobulins are usually heterotetrameric
glycoproteins of about 150,000 daltons, composed of two identical
light (L) chains and two identical heavy (H) chains. Each light
chain is linked to a heavy chain by one covalent disulfide bond,
while the number of disulfide linkages varies among the heavy
chains of different immunoglobulin isotypes. Each heavy and light
chain also has regularly spaced intrachain disulfide bridges. Each
heavy chain has at one end a variable domain (V.sub.H) followed by
a number of constant domains. Each light chain has a variable
domain at one end (V.sub.L) and a constant domain at its other end;
the constant domain of the light chain is aligned with the first
constant domain of the heavy chain, and the light- chain variable
domain is aligned with the variable domain of the heavy chain.
Particular amino acid residues are believed to form an interface
between the light- and heavy-chain variable domains.
[0049] The main Ig isotypes (classes) found in serum, and the
corresponding Ig heavy chains, shown in parentheses, are listed
below:
[0050] IgG (.gamma. chain): the principal Ig in serum, the main
antibody raised in response to an antigen, this antibody crosses
the placenta;
[0051] IgE (.epsilon. chain): this Ig binds tightly to mast cells
and basophils, and when additionally bound to antigen, causes
release of histamine and other mediators of immediate
hypersensitivity; plays a primary role in allergic reactions,
including hay fever, asthma and anaphylaxis; and may serve a
protective role against parasites;
[0052] IgA (.alpha. chain): this Ig is present in external
secretions, such as saliva, tears, mucous, and colostrum;
[0053] IgM (.mu. chain): the Ig first induced in response to an
antigen; it typically has lower affinity than other antibody
isotypes produced later and is typically pentameric.
[0054] IgD (.delta. chain): this Ig is found in relatively high
concentrations in umbilical cord blood, may be an early cell
receptor for antigen, and is the main lymphocyte cell surface
molecule.
[0055] The term "antibody" herein is used in the broadest sense and
specifically covers monoclonal antibodies (including, but not
limited to, full length monoclonal antibodies), polyclonal
antibodies, multispecific antibodies (e.g., bispecific antibodies),
and antibody fragments as long as they exhibit the desired
biological activity.
[0056] "Antibody fragments" comprise a portion of a full length
antibody, generally the antigen binding or variable (V) domain.
Examples of antibody fragments include Fab, Fab', F(ab').sub.2, and
Fv fragments; diabodies; linear antibodies; single-chain antibody
molecules; and multispecific antibodies formed from antibody
fragments.
[0057] 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 except for possible naturally occurring
mutations that may be present in minor amounts. Monoclonal
antibodies are highly specific, being directed against a single
antigenic site. Furthermore, in contrast to conventional
(polyclonal) antibody preparations which typically include
different antibodies directed against different determinants
(epitopes), each monoclonal antibody is directed against a single
determinant on the antigen.
[0058] The monoclonal antibodies herein specifically include
"chimeric" antibodies (immunoglobulins), as well as fragments of
such antibodies, as long as they exhibit the desired biological
activity (U.S. Pat. No. 4,816,567; Morrison et al., Proc. Natl.
Acad. Sci. USA 81:6851-6855 (1984); Oi et al., Biotechologies
4(3):214-221 (1986); and Liu et al., Proc. Natl. Acad Sci. USA
84:3439-43 (1987)).
[0059] "Humanized" or "CDR grafted" forms of non-human (e.g.,
murine) antibodies are human immunoglobulins (recipient antibody)
in which hypervariable region residues of the recipient are
replaced by hypervariable region residues from a non-human species
(donor antibody) such as mouse, rat, rabbit or nonhuman primate
having the desired specificity, affinity, and capacity. In some
instances, framework region (FR) residues of the human
immunoglobulin are also replaced by corresponding non-human
residues (so called "back mutations"). Furthermore, humanized
antibodies may be modified to comprise residues which are not found
in the recipient antibody or in the donor antibody, in order to
further improve antibody properties, such as affinity. In general,
the humanized antibody will comprise substantially all of at least
one, and typically two, variable domains, in which all or
substantially all of the hypervariable regions correspond to those
of a non-human immunoglobulin and all or substantially all of the
FRs are those of a human immunoglobulin sequence. The humanized
antibody optionally also will comprise at least a portion of an
immunoglobulin constant region (Fc), typically that of a human
immunoglobulin. For further details, see Jones et al., Nature
321:522-525 (1986); and Reichmann et al., Nature 332:323-329
(1988).
[0060] "Single-chain Fv" or "sFv" antibody fragments comprise the
V.sub.H and V.sub.L domains of antibody, wherein these domains are
present in a single polypeptide chain. Generally, the Fv
polypeptide further comprises a polypeptide linker between the
V.sub.H and V.sub.L domains which enables the sFv to form the
desired structure for antigen binding. For a review of sFv see
Pl{overscore (u)}ckthun in The Pharmacology of Monoclonal
Antibodies, vol. 113, Rosenburg and Moore eds. Springer-Verlag, New
York, pp. 269-315 (1994).
[0061] The term "diabodies" refers to small antibody fragments with
two antigen-binding sites, which fragments comprise a heavy chain
variable domain (V.sub.H) connected to a light chain variable
domain (V.sub.L) in the same polypeptide chain (V.sub.H-V.sub.L).
By using a linker that is too short to allow pairing between the
two domains on the same chain, the domains are forced to pair with
the complementary domains of another chain and create two
antigen-binding sites. Diabodies are described more fully in, for
example, EP 404,097; WO 93/11161; and Hollinger et al., Proc. Natl.
Acad. Sci. USA 90:6444-6448 (1993).
[0062] The expression "linear antibodies" when used throughout this
application refers to the antibodies described in Zapata et al.
Protein Eng. 8(10): 1057-1062 (1995). Briefly, these antibodies
comprise a pair of tandem Fd segments
(V.sub.H-C.sub.H1-V.sub.H-C.sub.H1) which form a pair of antigen
binding regions. Linear antibodies can be bispecific or
monospecific.
[0063] Antibodies of the IgG, IgE, IgA, IgM, and IgD isotypes may
have the same variable regions, i.e. the same antigen binding
cavities, even though they differ in the constant region of their
heavy chains. The constant regions of an immunoglobulin, e.g.
antibody are not involved directly in binding the antibody to an
antigen, but exhibit various effector functions, such as
participation of the antibody in antibody-dependent cellular
toxicity (ADCC).
[0064] Some of the main antibody isotypes (classes) are divided
into further sub-classes. IgG has four known subclasses: IgG1
(.gamma.1), IgG2 (.gamma.2), IgG3 (.gamma.3), and IgG4 (.gamma.4),
while IgA has two known sub-classes: IgA1 (.alpha.1) and IgA2
(.alpha.2).
[0065] The term "epitope" is used to refer to binding sites for
(monoclonal or polyclonal) antibodies on protein antigens.
[0066] Antibodies which bind to domain 1 and/or 2 within the amino
acid sequence of native sequence human CD22, or to essentially the
same epitope(s) bound by any of the monoclonal antibodies
specifically disclosed herein, such as HB22-7, HB22-23, and
HB22-33, can be identified by "epitope mapping." There are many
methods known in the art for mapping and characterizing the
location of epitopes on proteins, including solving the crystal
structure of an antibody-antigen complex, competition assays, gene
fragment expression assays, and synthetic peptide-based assays, as
described, for example, in Chapter 11 of Harlow and Lane, Using
Antibodies, a Laboratory Manual, Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y., 1999. According to the gene
fragment expression assays, the open reading frame encoding the
protein is fragmented either randomly or by specific genetic
constructions and the reactivity of the expressed fragments of the
protein with the antibody to be tested is determined. The gene
fragments may, for example, be produced by PCR and then transcribed
and translated into protein in vitro, in the presence of
radioactive amino acids. The binding of the antibody to the
radioactively labeled protein fragments is then determined by
immunoprecipitation and gel electrophoresis. Certain epitopes can
also be identified by using large libraries of random peptide
sequences displayed on the surface of phage particles (phage
libraries). Alternatively, a defined library of overlapping peptide
fragments can be tested for binding to the test antibody in simple
binding assays. The latter approach is suitable to define linear
epitopes of about 5 to 15 amino acids.
[0067] An antibody binds "essentially the same epitope" as a
reference antibody, when the two antibodies recognize identical or
sterically overlapping epitopes. The most widely used and rapid
methods for determining whether two epitopes bind to identical or
sterically overlapping epitopes are competition assays (e.g.
competition ELISA assays), which can be configured in all number of
different formats, using either labeled antigen or labeled
antibody. Usually, the antigen is immobilized on a 96-well plate,
and the ability of unlabeled antibodies to block the binding of
labeled antibodies is measured using radioactive or enzyme
labels.
[0068] The term amino acid or amino acid residue, as used herein,
refers to naturally occurring L amino acids or to D amino acids as
described further below with respect to variants. The commonly used
one- and three-letter abbreviations for amino acids are used herein
(Bruce Alberts et al., Molecular Biology of the Cell, Garland
Publishing, Inc., New York (3d ed. 1994)).
[0069] As used herein, the term "polypeptide" encompasses peptides
and proteins, including fusion proteins.
[0070] "Sequence identity" is defined as the percentage of amino
acid residues in a candidate sequence that are identical with the
amino acid residues in a native 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.
The % sequence identity values can be generated by the NCBI
BLAST2.0 software as defined by Altschul et al., (1997), "Gapped
BLAST and PSI-BLAST: a new generation of protein database search
programs", Nucleic Acids Res., 25:3389-3402. The parameters are set
to default values, with the exception of the Penalty for mismatch,
which is set to -1.
[0071] As used herein, "treatment" or "treating" is an approach for
obtaining beneficial or desired clinical results. For purposes of
this invention, beneficial or desired clinical results include, but
are not limited to, alleviation of symptoms, diminishment of extent
of disease, stabilized (i.e., not worsening) state of disease,
delay or slowing of disease progression, amelioration or palliation
of the disease state, and remission (whether partial or total),
whether detectable or undetectable. "Treatment" or "treating" can
also mean prolonging survival as compared to expected survival if
not receiving treatment. "Treatment" or "treating" is an
intervention performed with the intention of preventing the
development or altering the pathology of a disorder. Accordingly,
"treatment" or "treating" refers to both therapeutic treatment and
prophylactic or preventative measures. Those in need of treatment
include those already with the disorder as well as those in which
the disorder is to be prevented. With respect to autoimmune
disease, the treatment results in some improvement, amelioration,
stabilization and/or delay in at least one clinical symptom of the
autoimmune disease in the subject. In the context of B cell
malignancies, the treatment may reduce the number of malignant
cells; reduce the tumor size; inhibit (slow down or stop) the
spread of malignant cells, including infiltration into peripheral
organs, e.g. soft tissue or bone; inhibit (slow down or stop)
metastasis; inhibit tumor growth; provide relief from symptoms
associated with a B cell malignancy; reduce mortality; improve
quality of life, etc. Treatment with the antibodies herein may
result in cytostatic and/or cytotoxic effects.
[0072] The term "B cell malignancy," and grammatical variants
thereof, are used in the broadest sense to refer to malignancies or
neoplasms of B cells that typically arise in lymphoid tissues, such
as bone marrow or lymph nodes, but may also arise in non-lymphoid
tissues, such as thyroid, gastrointestinal tract, salivary gland
and conjunctiva. The treatment methods of the present invention
specifically concern CD22-positive B cell malignancies including,
without limitation, B-cell subtype of non-Hodgkin's lymphoma,
Burkitt's lymphoma, multiple myeloma, chronic lymphocytic leukemia,
hairy cell leukemia, and prolymphocytic leukemia.
[0073] The term "autoimmune disease" refers to a condition which
results from, or is aggravated by, the production of antibodies
reactive with normal body tissues. It is a condition in which the
immune system mistakenly attacks the body's own organs and
tissues.
B. Detailed Description
1. Antibodies
[0074] Blocking anti-CD22 monoclonal antibodies designated HB22-7,
HB22-23, HB22-33, HB22-5, HB22-13, and HB22-196 are known, and have
been disclosed in U.S. Pat. No. 5,484,892, Tuscano et al., Eur. J.
Immunol. 26:1246 (1996), and Tuscano et al., Blood 94(4), 1382-1392
(1999). HB22-7 and HB22-23 are available from the American Type
Culture Collection (ATCC), 12302 Parklawn Drive, Rockville, Md.
20852, under Accession Nos. HB22347 and HB11349, respectively. The
preparation of these antibodies is also described in Example 1
below. Epitope mapping of CD22 has shown that these blocking
monoclonal antibodies bind to the first two Ig-like domains or to
epitopes which are associated with the first two Ig-like domains of
human CD22 (U.S. Pat. No. 5,484,892 and Tedder et al., Annu. Rev.
Immunol. 15:481-504 (1997)). The heavy and light chain variable
region sequences of the antibodies are also disclosed in the
present application.
[0075] The present invention is based, in part, on the unexpectedly
superior properties of blocking anti-CD22 antibodies having the
overall characteristics of HB22-7, HB22-23, HB22-33, HB22-5,
HB22-13, and HB22-196 in the treatment of B-cell malignancies,
based on results obtained in a xenograft model of B-cell type
non-Hodgkin's lymphoma (NHL). The invention is further based on the
use of blocking anti-CD22 antibodies having the overall
characteristics of HB22-7, HB22-23, HB22-33, HB22-5, HB22-13, and
HB22-196 in the treatment of autoimmune disease.
[0076] The anti-CD22 monoclonal antibodies can be made by any
standard method known in the art, such as, for example, by the
hybridoma method (Koehler and Milstein, Nature 256:495-497 (1975);
and Goding, Monoclonal Antibodies: Principles and Practice,
pp.59-103, (Academic Press, 1986)), or by recombinant techniques,
disclosed, for example, in U.S. Pat. No. 4,816,567, and by Wood et
al., Nature 314:446-9 (1985).
[0077] It is now also possible to produce transgenic animals (e.g.
mice) that are capable, upon immunization, of producing a
repertoire of human antibodies in the absence of endogenous
immunoglobulin production. For example, it has been described that
the homozygous deletion of the antibody heavy chain joining region
(J.sub.H) gene in chimeric and germ-line mutant mice results in
complete inhibition of endogenous antibody production. Transfer of
the human germ-line immunoglobulin gene array in such germ-line
mutant mice will result in the production of human antibodies upon
antigen challenge. See, e.g. Jakobovits et al., Proc. Natl. Acad.
Sci. USA 90, 2551-255 (1993); Jakobovits et al., Nature 362,
255-258 (1993).
[0078] Mendez et al. (Nature Genetics 15: 146-156 (1997)) have
further improved the technology and have generated a line of
transgenic mice designated as "Xenomouse II" that, when challenged
with an antigen, generates high affinity fully human antibodies.
This was achieved by germ-line integration of megabase human heavy
chain and light chain loci into mice with deletions in the
endogenous J.sub.H segment as described above. The Xenomouse II
harbors 1,020 kb of human heavy chain locus containing
approximately 66 V.sub.H genes, complete D.sub.H and J.sub.H
regions and three different constant regions (.mu., .delta. and
.chi.), and also harbors 800 kb of human .kappa. locus containing
32 V.kappa. genes, J.kappa. segments and C.kappa. genes. The
antibodies produced in these mice closely resemble those seen in
humans in all respects, including gene rearrangement, assembly, and
repertoire. The human antibodies are preferentially expressed over
endogenous antibodies due to deletions in the endogenous J.sub.H
segment that prevents gene rearrangement in the murine locus.
[0079] Alternatively, phage display technology (McCafferty et al.,
Nature 348, 552-553 (1990)) can be used to produce human antibodies
and antibody fragments in vitro, from immunoglobulin variable (V)
domain gene repertoires from unimmunized donors. According to this
technique, antibody V domain genes are cloned in-frame into either
a major or minor coat protein gene of a filamentous bacteriophage,
such as M13 or fd, and displayed as functional antibody fragments
on the surface of the phage particle. Because the filamentous
particle contains a single-stranded DNA copy of the phage genome,
selections based on the functional properties of the antibody also
result in selection of the gene encoding the antibody exhibiting
those properties. Thus, the phage mimics some of the properties of
the B-cell. Phage display can be performed in a variety of formats;
for their review see, e.g. Johnson, Kevin S. and Chiswell, David
J., Current Opinion in Structural Biology 3, 564-571 (1993).
Several sources of V-gene segments can be used for phage display.
Clackson et al., Nature 352, 624-628 (1991) isolated a diverse
array of anti-oxazolone antibodies from a small random
combinatorial library of V-genes derived from the spleens of
immunized mice. A repertoire of V-genes from unimmunized human
donors can be constructed and antibodies to a diverse array of
antigens (including self-antigens) can be isolated essentially
following the techniques described by Marks et al., J. Mol. Biol
222, 581-597 (1991), or Griffith et al., EMBO J. 12, 725-734
(1993). In a natural immune response, antibody genes accumulate
mutations at a high rate (somatic hypermutation). Some of the
changes introduced will confer higher affinity, and B cells
displaying high-affinity surface immunoglobulin are preferentially
replicated and differentiated during subsequent antigen challenge.
This natural process can be mimicked by employing the technique
known as "chain shuffling" (Marks et al., Bio/Technol. 10, 779-783
0077]). In this method, the affinity of "primary" human antibodies
obtained by phage display can be improved by sequentially replacing
the heavy and light chain V-region genes with repertoires of
naturally occurring variants (repertoires) of V-domain genes
obtained from unimmunized donors. This techniques allows the
production of antibodies and antibody fragments with affinities in
the nM range. A strategy for making very large phage antibody
repertoires has been described by Waterhouse et al., Nucl. Acids
Res. 21, 2265-2266 (1993).
[0080] For further information concerning the production of
monoclonal antibodies see also Goding, J. W., Monoclonal
Antibodies: Principles and Practice, 3rd Edition, Academic Press,
Inc., London, San Diego, 1996; Liddell and Weeks: Antibody
Technology: A Comprehensive Overview, Bios Scientific Publishers:
Oxford, UK, 1995; Breitling and Dubel: Recombinant Antibodies, John
Wiley & Sons, New York, 1999; and Phage Display: A Laboratory
Manual, Barbas et al., editors, Cold Springs Harbor Laboratory,
Cold Spring Harbor, 2001.
[0081] Various techniques have been developed for the production of
antibody fragments. Traditionally, these fragments were derived via
proteolytic digestion of intact antibodies (see, e.g., Morimoto et
al., J. Biochem. Biophys. Methods 24:107-117 (1992) and Brennan et
al., Science 229:81 (1985)). However, these fragments can now be
produced directly by recombinant host cells. For example, Fab'-SH
fragments can be directly recovered from E. coli and chemically
coupled to form F(ab').sub.2 fragments (Carter et al.,
Bio/Technology 10:163-167 (1992)). In another embodiment, the
F(ab').sub.2 is formed using the leucine zipper GCN4 to promote
assembly of the F(ab').sub.2 molecule. According to another
approach, Fv, Fab or F(ab').sub.2 fragments can be isolated
directly from recombinant host cell culture. Other techniques for
the production of antibody fragments will be apparent to the
skilled practitioner.
[0082] Heteroconjugate antibodies, composed of two covalently
joined antibodies, are also within the scope of the present
invention. Such antibodies have, for example, been proposed to
target immune system cells to unwanted cells (U.S. Pat. No.
4,676,980), and for treatment of HIV infection (PCT application
publication Nos. WO 91/00360 and WO 92/200373). Heteroconjugate
antibodies may be made using any convenient cross-linking methods,
using well known, commercially available cross-linking agents.
[0083] The antibodies of the present invention, whether rodent,
human, or humanized may also have a further antigen-specificity, to
form bispecific antibodies. The second binding specificity may be
directed, for example, against a further B cell antigen, such as
CD19, CD20, CD52, and other CD antigens expressed on B cells,
especially antigens associated with the targeted B cell malignancy.
For example, CD20 is known to be expressed in more than 90% of
non-Hodgkin's lymphomas. An anti-CD20 antibody (Rituxan.RTM., IDEC
Pharmaceuticals) is in clinical use for the treatment of
non-Hodgkin's lymphoma. CAMPATH-1H (anti-CD52w) is another antibody
developed for treating B cell malignancies. Bispecific antibodies
including a binding specificity to the CD20 or CD52 antigen are
specifically included within the scope herein. Another B cell
antigen to which the bispecific antibodies of the present invention
can bind is HLA-DR10 (Lym-1), a known marker of non-Hodgkin's
lymphoma. Bispecific antibodies can be generated to enhance tumor
localization as well as to recruit and/or augment the
tumor-specific immune response. Examples of other antigen targets
include, CD3, CD28, and the Fc receptors (CD16, CD64 and CD89).
Bispecific antibodies are expected to have enhanced cytotoxicity
and, as a result, improved remission rate and survival.
[0084] Antibodies binding to essentially the same epitope as
HB22-7, HB22-23, HB22-33, HB22-5, HB22-13, and/or HB22-196 can be
identified by epitope mapping. The simplest way to determine
whether two different antibodies recognize the same epitope is a
competition binding assay. This method determines if the antibodies
are able to block each other's binding to the antigen, and works
for both conformational and linear epitopes. The competition
binding assay can be configured in a large number of different
formats using either labeled antigen or labeled antibody. In the
most common version of this assay, the antigen is immobilized on a
96-well plate. The ability of unlabeled antibodies to block the
binding of labeled antibodies to the antigen is then measured using
radioactive or enzyme labels. For further details see, for example,
Wagener et al., J. Immunol., 130:2308-2315 (1983); Wagener et al.,
J. Immunol. Methods, 68:269-274 (1984); Kuroki et al., Cancer Res.
50:4872-4879 (1990); Kuroki et al., Immunol. Invest. 21:523-538
(1992); Kuroki et al., Hybridoma 11:391-407 (1992), and Using
Antibodies: A Laboratory Manual, Ed Harlow and David Lane editors,
Cold Springs Harbor Laboratory Press, Cold Springs Harbor, N.Y.,
1999, pp. 386-389.
[0085] Alternatively, or in addition, epitope mapping can be
performed by using a technique based on fragmentation of the
antigen to which the antibody binds, either randomly or by specific
genetic construction, and determining the reactivity of the
fragments obtained with the antibody. Fragmentation can also be
performed on the nucleic acid level, for example by PCR technique,
followed by transcription and translation into protein in vitro in
the presence of radioactive amino acids. For further details see,
for example, Harlow and Lane, supra, pp. 390-392.
[0086] According to a further method of epitope mapping, a set of
overlapping peptides is synthesized, each corresponding to a small
linear segment of the protein antigen, and arrayed on a solid
phase. The panel of peptides is then probed with the test antibody,
and bound antibody is detected using an enzyme-labeled secondary
antibody. (Harlow and Lane, supra, pp. 393-396.)
[0087] An additional method well known in the art for epitope
mapping is antibody selection from a random synthetic or a phage
display peptide library. Phage display libraries are constructed by
cloning complex mixtures of peptide-encoding oligonucleotides into
the amino terminus of the minor coat protein gene of the f1-type
ssDNA phage. Such phage display libraries are commercially
available, for example, from New England Biolabs. The libraries are
amplified as stocks, and then an aliquot sufficient to represent
multiple copies of each independent clone is mixed with the
antibody of interest. Antibody-bound phage are collected by a
procedure called "biopanning," and unbound phage are removed. The
bound phage are eluted and used to infect bacteria, and the
selected stock is amplified. Individual plaques of the final
selected stock are growth and checked for specific antibody
reactivity, e.g. by ELISA, and the DNA around the insert site is
sequenced. Analysis of the sequence encoding the peptide to which
the antibody binds defines the specificity of the antibody. For
further details see, e.g. Smith and Scott, Methods Enzymol.
217:228-257 (1993), and Harlow and Lane, supra, pp. 397-398.
[0088] Non-human (rodent) antibodies can be further modified to
make them more suitable for human clinical application. Chimeric
antibodies are produced with mouse variable region gene segments of
desired specificity spliced into human constant domain gene
segments (see, e.g. U.S. Pat. No. 4,816,567).
[0089] Non-human (rodent) antibodies can also be humanized in order
to avoid issues of antigenicity when using the antibodies in human
therapy. Generally, a humanized antibody has one or more amino acid
residues introduced into it from a non-human source. These
non-human amino acid residues are often referred to as "import"
residues, which are typically taken from an "import" variable
domain. Humanization can be essentially performed following the
method of Winter and co-workers (Jones et al., Nature, 321:522-525
(1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et
al., Science, 239:1534-1536 (1988)), by substituting rodent CDRs or
CDR sequences for the corresponding sequences of a human antibody.
Despite the relatively straightforward nature of antibody
humanization, simple grafting of the rodent CDR's into human
frameworks (FR) does not always reconstitute the binding affinity
and specificity of the original rodent monoclonal antibody.
Properties of a humanized antibody can be improved by suitable
design, including, for example, substitution of residues from the
rodent antibody into the human framework (backmutations). The
positions for such backmutations can be determined by sequence and
structural analysis, or by analysis of the variable regions'
three-dimensional model. In addition, phage display libraries can
be used to vary amino acids at chosen positions within the antibody
sequence. The properties of a humanized antibody are also affected
by the choice of the human framework. Early experiments used a
limited subset of well-characterized human monoclonal antibodies,
irrespective of the sequence identity to the rodent monoclonal
antibody (the so-called fixed frameworks approach). More recently,
some groups use variable regions with high amino acid sequence
identity to the rodent variable regions (homology matching or
best-fit method). According to another approach, consensus or
germline sequences are used, or fragments of the framework
sequences within each light or heavy chain variable region are
selected from several different human monoclonal antibodies.
[0090] Amino acid variants of antibodies prepared by any technique
discussed above or otherwise available can be prepared by
introducing appropriate nucleotide changes into the anti-CD22 DNA,
or, for example, by peptide synthesis. The amino acid changes also
may alter post-translational processes of the humanized or variant
anti-CD22 antibody, such as changing the number or position of
glycosylation sites.
[0091] Antibodies are glycosylated at conserved positions in their
constant regions (Jefferis and Lund, Chem. Immunol. 65:111-128
(1997); Wright and Morrison, TibTECH 15:26-32 (1997)). The
oligosaccharide side chains of the immunoglobulins affect the
protein's function (Boyd et al., Mol. Immunol. 32:1311-1318 (1996);
Wittwe and Howard, Biochem. 29:4175-4180 (1990)), and the
intramolecular interaction between portions of the glycoprotein
which can affect the conformation and presented three-dimensional
surface of the glycoprotein (Jefferis and Lund, supra; Wyss and
Wagner, Current Opin. Biotech. 7:409-416 (1996)). Oligosaccharides
may also serve to target a given glycoprotein to certain molecules
based upon specific recognition structures. For example, it has
been reported that in agalactosylated IgG, the oligosaccharide
moiety `flips` out of the inter-CH2 space and terminal
N-acetylglucosamine residues become available to bind mannose
binding protein (Malhotra et al., Nature Med. 1:237-243 (1995)).
Removal by glycopeptidase of the oligosaccharides from CAMPATH-1H
(a recombinant humanized murine monoclonal IgG1 antibody which
recognizes the CDw52 antigen of human lymphocytes) produced in
Chinese Hamster Ovary (CHO) cells resulted in a complete reduction
in complement mediated lysis (CMCL) (Boyd et al., Mol. Immunol.
32:1311-1318 (1996)), while selective removal of sialic acid
residues using neuraminidase resulted in no loss of CMCL.
Glycosylation of antibodies has also been reported to affect
antibody-dependent cellular cytotoxicity (ADCC). In particular, CHO
cells with tetracycline-regulated expression of
.beta.(1,4)-N-acetylglucosaminyltran- sferase III (GnTIII), a
glycosyltransferase catalyzing formation of bisecting GlcNAc, was
reported to have improved ADCC activity (Umana et al., Mature
Biotech. 17:176-180 (1999)).
[0092] Glycosylation variants of antibodies can be prepared by
modifying the glycosylation sites in the underlying nucleotide
sequence. In addition, the glycosylation of antibodies may also be
altered without altering the underlying nucleotide sequence.
Glycosylation largely depends on the host cell used to express the
antibody. Since the cell type used for expression of recombinant
glycoproteins, e.g. antibodies, as potential therapeutics is rarely
the native cell, significant variations in the glycosylation
pattern of the antibodies can be expected (see, e.g. Hse et al., J.
Biol. Chem. 272:9062-9070 (1997)). In addition to the choice of
host cells, factors which affect glycosylation during recombinant
production of antibodies include growth mode, media formulation,
culture density, oxygenation, pH, purification schemes and the
like. Various methods have been proposed to alter the glycosylation
pattern achieved in a particular host organism including
introducing or overexpressing certain enzymes involved in
oligosaccharide production (U.S. Pat. Nos. 5,047,335; 5,510,261 and
5.278,299). Glycosylation, or certain types of glycosylation, can
be enzymatically removed from the glycoprotein, for example using
endoglycosidase H (Endo H). In addition, the recombinant host cell
can be genetically engineered, e.g. made defective in processing
certain types of polysaccharides. These and similar techniques are
well known in the art.
[0093] The antibodies of the present invention may also be used by
the antibody-directed enzyme prodrug therapy (ADEPT). ADEPT is a
technology that utilizes the specificity of monoclonal antibodies
targeting tumor antigens to target catalytic enzymes to the surface
of cancer cells. There, the enzymes are in position to activate
prodrug forms (e.g., a peptidyl chemotherapeutic agent, see
WO81/01145) of anti-cancer drugs to their fully active form. See,
for example, WO 88/07378 and U.S. Pat. No. 4,975,278.
[0094] Enzymes that are useful in the method of this invention
include, but are not limited to, alkaline phosphatase useful for
converting phosphate-containing prodrugs into free drugs;
arylsulfatase useful for converting sulfate-containing prodrugs
into free drugs; cytosine deaminase useful for converting non-toxic
5-fluorocytosine into the anti-cancer drug, 5-fluorouracil;
proteases, such as serratia protease, thermolysin, subtilisin,
carboxypeptidases and cathepsins (such as cathepsins B and L), that
are useful for converting peptide-containing prodrugs into free
drugs; D-alanylcarboxypeptidases, useful for converting prodrugs
that contain D-amino acid substituents; carbohydrate-cleaving
enzymes such as .beta.-galactosidase and neuraminidase useful for
converting glycosylated prodrugs into free drugs; .beta.-lactamase
useful for converting drugs derivatized with .beta.-lactams into
free drugs; and penicillin amidases, such as penicillin V amidase
or penicillin G amidase, useful for converting drugs derivatized at
their amine nitrogens with phenoxyacetyl or phenylacetyl groups,
respectively, into free drugs. Alternatively, antibodies with
enzymatic activity, also known in the art as "abzymes", can be used
to convert the prodrugs of the invention into free active drugs
(see, e.g., Massey, Nature 328:457-458 (1987)). Antibody-abzyme
conjugates can be prepared as described herein for delivery of the
abzyme to a tumor cell population.
[0095] Immunoconjugates of the antibodies herein are also
specifically encompassed by this invention. Immunoconjugates
comprise an antibody conjugated to a cytotoxic agent, such as
chemotherapeutic agent, a toxin, or a radioisotope.
[0096] Specifically, the efficacy of the anti-CD22 antibodies
herein can be further enhanced by conjugation to a cytotoxic
radioisotope, to allow targeting a radiotherapy specifically to
target sites (radioimmunotherapy). Suitable radioisotopes include,
for example, I.sup.131 and Y.sup.90, both used in clinical
practice. Other suitable radioisotopes include, without limitation,
In.sup.111, Cu.sup.67, I.sup.131, As.sup.211, Bi.sup.212,
Bi.sup.213, and Re.sup.186.
[0097] Chemotherapeutic agents useful in the generation of
immunoconjugates include, for example, adriamycin, doxorubicin,
epirubicin, 5-fluorouracil, cytosine arabinoside ("Ara-C"),
cyclophosphamide, thiotepa, busulfan, cytoxin, taxoids, e.g.,
paclitaxel (Taxol, Bristol-Myers Squibb Oncology, Princeton, N.J.),
and doxetaxel (Taxotere, Rhne-Poulenc Rorer, Antony, Rnace),
toxotere, methotrexate, cisplatin, melphalan, vinblastine,
bleomycin, etoposide, ifosfamide, mitomycin C, mitoxantrone,
vincristine, vinorelbine, carboplatin, teniposide, daunomycin,
carminomycin, aminopterin, dactinomycin, mitomycins, esperamicins
(see U.S. Pat. No. 4,675,187), 5-FU, 6-thioguanine,
6-mercaptopurine, actinomycin D, VP-16, chlorambucil, melphalan,
and other related nitrogen mustards.
[0098] Toxins to be used in the immunoconjugates herein include,
for example, diphtheria A chain, exotoxin A chain, ricin A chain,
enomycin, and tricothecenes. Specifically included are
antibody-maytansinoid and antibody-calicheamicin conjugates.
Immunoconjugates containing maytansinoids are disclosed, for
example, in U.S. Pat. Nos. 5,208,020, 5,416,020 and European Patent
EP 0 425 235. See also Liu et al., Proc. Natl. Acad. Sci. USA
93:8618-8623 (1996). Antibody-calicheamicin conjugates are
disclosed, e.g. in 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.
[0099] Conjugates of the antibody and cytotoxic agent are made
using a variety of bifunctional protein coupling agents such as
N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP),
iminothiolane (IT), bifunctional derivatives of imidoesters (such
as dimethyl adipimidate HCl), active esters (such as disuccinimidyl
suberate), aldehydes (such as glutareldehyde), bis-azido compounds
(such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium
derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine),
diisocyanates (such as tolyene 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, WO 94/11026.
[0100] Covalent modifications of the anti-CD22 antibodies are also
included within the scope of this invention. They may be made by
chemical synthesis or by enzymatic or chemical cleavage of the
antibody, if applicable. Other types of covalent modifications of
the antibody are introduced into the molecule by reacting targeted
amino acid residues of the antibody with an organic derivatizing
agent that is capable of reacting with selected side chains or the
N- or C-terminal residues. A preferred type of covalent
modification of the antibodies comprises linking the antibodies to
one of a variety of nonproteinaceous polymers, e.g., polyethylene
glycol, polypropylene glycol, or polyoxyalkylenes, in the manner
well known in the art.
2. Pharmaceutical Formulations and Treatment Methods
[0101] B-cell type Non-Hodgkin's Lymphoma is a term that is used to
encompass a large group (over 29 types) of lymphomas caused by
malignant (cancerous) B cell lymphocytes, and represents a large
subset of the known types of lymphoma. B-cells are known to undergo
many changes in their life cycle dependent on complex intracellular
signaling processes, and apparently different types of B-cell
malignancies can occur at different stages of the life cycle of
B-cells. At the stem cell stage, acute lymphocytic leukemia (ALL)
or lymphoblastic lymphoma/leukemia can typically develop. Precursor
B-cells can develop precursor B lymphoblastic lymphoma/leukemia.
Typical malignancies of immature B-cells include small non-cleaved
cell lymphoma and possibly Burkitt's/non-Burkitt's lymphoma. B
cells before antigen exposure typically develop chronic lymphocytic
leukemia (CLL) or small lymphocytic lymphoma, while after antigen
exposure typically follicular lymphomas, large cell lymphoma and
immunoblastic lymphoma are observed. There are also classification
systems that characterize B-cell lymphomas by the rate of growth
distinguishing aggressive (fast growing) and indolent (slow
growing) lymphomas. For example, Burkitt's/non-Burkitt's lymphoma
and LCL lymphoma belong in the aggressive group, while indolent
lymphomas include follicular center cell lymphomas (FCCL),
follicular large cell lymphomas, and follicular small cleaved cell
lymphomas.
[0102] Non-Hodgkin's Lymphomas are also characterized by the stage
of development. Stage I: cancer is found in only one lymph node
area, or in only one area or organ outside the lymph nodes. Stage
II: (1) Cancer is found in two or more lymph node areas on the same
side of the diaphragm (the thin muscle under the lungs that helps
breathing), or, (2) cancer is found in only one area or organ
outside the lymph nodes and in the lymph nodes around it, or (3)
other lymph node areas on the same side of the diaphragm may also
have cancer. Stage III: Cancer is found in lymph node areas on both
sides of the diaphragm. The cancer may also have spread to an area
or organ near the lymph node areas and/or to the spleen. Stage IV:
(1) Cancer has spread to more than one organ or organs outside the
lymph system; cancer cells may or may not be found in the lymph
nodes near these organs, or (2) cancer has spread to only one organ
outside the lymph system, but lymph nodes far away from that organ
are involved.
[0103] Current treatment options of B-cell malignancies, including
non-Hodgkin's lymphomas depend on the type and stage of malignancy.
Typical treatment regimens include radiation therapy, also referred
to as external beam therapy, chemotherapy, immunotherapy, and
combinations of these approaches. One promising approach is
radioimmunotherapy (RIT). With external beam therapy, a limited
area of the body is irradiated. With chemotherapy, the treatment is
systemic, and often adversely affects normal cells, causing severe
toxic side-effects. Targeted RIT is an approach in which a B-cell
specific antibody delivers a toxic substance to the site of tumor.
The therapeutic potential of RIT in patients with B-cell NHL has
been shown using different targets, including CD20, CD19, CD22, and
HLA-DR10 (Lym-1). More recently, combined modality therapy (CMT)
has become an increasingly frequent maneuver for the treatment of
solid tumors, and includes radiosensitization of cancer cells by
drugs, and the direct cytotoxic effect of chemotherapy. The most
common chemotherapy regiment for treating NHL is
Cyclophosphamide-Hydroxydoxorub- icin-Oncovin
(vincristine)-Prednisone (CHOP) combination therapy. A randomized
study of aggressive, but early stage NHL showed superior results
with CHOP plus involved field radiation over treatment with CHOP
alone. Despite its promise, the disadvantage of treatments
involving external beam radiation is that external beam radiation
can only be delivered in high doses to a limited region of the
body, while NHL is mostly widespread. Accordingly, CMT has proven
clinically useful for locally advanced malignancies.
[0104] Another current approach is combined modality
radioimmunotherapy (CMRIT), which pairs the specific delivery of
systemic radiation (e.g. .sup.90Y-DOTA-peptide-Lym-1) to NHL with
the systemic radiation sensitizing effects of an additional
chemotherapeutic agent. Because in CMRIT radiation is delivered
continuously, cancer cells that are hypoxic may re-oxygenate, or
pass through the radiosensitive G.sub.2/M phase of the cell cycle
during the course of treatment, making cure more likely. In
addition, CMRIT provides specificity first, by the specific
targeting of NHL by Lym-1, and second by timing. This allows the
radiation sensitizer to potentially synergize only at the sites
targeted by RIT, thus maximizing efficacy and minimizing toxicity.
Several previous xenograft studies have demonstrated improved
synergy when the radiation synthesizer (Taxol) was given 24-48
hours after RIT.
[0105] Although CMRIT is currently viewed as the most advanced
therapeutic approach for the treatment of NHL, the antibodies of
the present invention alone have been demonstrated to provide
superior results both in terms of tumor volume reduction, cure rate
and overall survival, when tested in the well accepted Raji and
Ramos lymphoma xenograft models.
[0106] Autoimmune diseases are caused by a breakdown in
self-tolerance leading to subsequent immune responses against self,
including the production of autoantibodies and deposition of
immunoglobulin in affected tissues. Autoantibodies form immune
complexes that promote complement and Fc-receptor mediated tissue
inflammation and destruction. Since B cells are the source of
autoantibodies, they afford a rational target for treatment of
these types of immune-mediated diseases. B cells also can present
antigen and regulate the development of effector T cells. The
pathologic mechanisms of these diseases are complex and often
involve a combination of humoral and cellular immune
mechanisms.
[0107] Most autoimmune diseases result from, or are aggravated by,
the production of antibodies reactive with normal body tissues.
Antibodies are produced by B cells following antigen stimulation
and activation. Therefore, blocking CD22 function can inhibit the
production of antibodies, including autoreactive antibodies. More
than 80 autoimmune diseases have been identified. Autoimmune
diseases, their etiology and treatment are discussed extensively in
the Autoimmune Diseases Research Plan published by the Autoimmune
Diseases Coordinating Committee of the National Institutes of
Health. Autoimmune diseases that can be treated according to the
present invention include, but are not limited to immune complex
disorders such as those that result in glomerulonephritis,
Goodspature's syndrome, necrotizing vasculitis, lymphadenitis,
peri-arteritis nodosa and systemic lupus erythematosis. Other
illustrative autoimmune diseases include but are not limited to
rheumatoid arthritis, psoriatic arthritis, systemic lupus
erythematosis, psoriasis, ulcerative colitis, systemic sclerosis,
dermatomyositis/polymyositis, anti-phospholipid antibody syndrome,
scleroderma, perphigus vulgaris, ANCA-associated vasculitis (e.g.,
Wegener's granulomatosis, microscopic polyangiitis), urveitis,
Sjogren's syndrome, Crohn's disease, Reiter's syndrome, ankylosing
spondylitis, Lyme arthritis, Guillain-Barre syndrome, Hashimoto's
thyroiditis, and cardiomyopathy. Other diseases associated with
antibody production include, but are not limited to multiple
sclerosis, atopic dermatitis, thrombocytopenic purpura,
agranulocytosis, autoimmune hemolytic anemias, immune reactions
against foreign antigens such as fetal A-B-O blood groups during
pregnancy, myasthenia gravis, Type I diabetes, Graves' disease, and
allergic responses. The methods of the invention may be used to
treat any other disorder or condition in which B cells or
antibodies are implicated including, for example, transplant
rejection.
[0108] The anti-CD22 antibodies herein are typically administered
in the form of pharmaceutical formulations well known to all
pharmaceutical chemists. See, e.g. Remington's Pharmaceutical
Sciences, (15th Edition, Mack Publishing Company, Easton, Pa.
(1975)), particularly Chapter 87, by Blaug, Seymour. These
formulations include for example, powders, pastes, ointments,
jelly, waxes, oils, lipids, anhydrous absorption bases,
oil-in-water or water-in-oil emulsions, emulsions carbowax
(polyethylene glycols of a variety of molecular weights),
semi-solid gels, and semi-solid mixtures containing carbowax. A
typical dosage form is a sterile, isotonic, water-based solution
suitable for administration by the intravenous (i.v.) route. The
concentration of the antibodies of the invention in the
pharmaceutical formulations can vary widely, i.e., from less than
about 0.1%, usually at or at least about 2% to as much as 20% to
50% or more by weight, and will be selected primarily by fluid
volumes, viscosities, etc., in accordance with the particular mode
of administration selected.
[0109] The compositions of the invention may also be administered
via liposomes. Liposomes include emulsions, foams, micelles,
insoluble monolayers, liquid crystals, phospholipid dispersions,
lamellar layers and the like. In these preparations the composition
of the invention to be delivered is incorporated as part of a
liposome, alone or in conjunction with a molecule which binds to a
desired target, such as an antibody, or with other therapeutic or
immunogenic compositions. Liposomes for use in the invention are
formed from standard vesicle-forming lipids, which generally
include neutral and negatively charged phospholipids and a sterol,
such as cholesterol. The selection of lipids is generally guided by
consideration of, e.g., liposome size, acid lability and stability
of the liposomes in the blood stream. A variety of methods are
available for preparing liposomes, as described in, e.g., Szoka et
al. Ann. Rev. Biophys. Bioeng. 9:467 (1980), U.S. Pat. Nos.
4,235,871, 4,501,728, 4,837,028, and 5,019,369.
[0110] The antibodies of the present invention can be administered
alone or in combination with other therapeutic regimens. For
example, in the case of B-cell malignancies, such regimes or
therapies include chemotherapy, radioimmunotherapy (RIT),
chemotherapy and external beam radiation (combined modality
therapy, CMT), combined modality radioimmunotherapy (CMRIT), or
cytokines alone or in combination, etc. Thus, the anti-CD22
antibodies of the present invention can be combined with CHOP
(Cyclophosphamide-Hydroxydoxorubicin-Oncovin
(vincristine)-Prednisolone), the most common chemotherapy regimen
for treating non-Hodgkin's lymphoma. In addition, the anti-CD22
antibodies herein may be administered in combination with other
antibodies, including anti-CD19, anti-CD20 and other anti-CD22
antibodies, such as LymphoCide.TM. (Immunomedics, Inc.) or
LymphoCide Y-90. See, for example, Stein et al., Drugs of the
Future 18:997-1004 (1993); Behr et al., Clinical Cancer Research
5:3304s-33314s, 1999 (suppl.); Juweid et al., Cancer Res.
55:5899s-5907s, 1995; Behr et al., Tumor Targeting 3:32-40 (1998),
and U.S. Pat. Nos. 6,183,744, 6,187,287, and 6,254,868.
[0111] The inventive treatments may also be employed in combination
with other therapies for autoimmune disorders. In particular
embodiments, the subject is treated with the antibodies of the
invention as well as with an anti-CD20 antibody (e.g.,
Rituxan.RTM., IDEC Pharmaceuticals) and/or an anti-inflammatory
drug (e.g., corticosteroids).
[0112] In particular embodiments, the patients to be treated in
accordance with the present invention will have CD22 expressed on
their malignant B cells. The presence of the CD22 antigen can be
confirmed by standard techniques, such as immunohistochemistry,
FACS, binding assay with labeled (e.g. radiolabeled) anti-CD22
antibody.
[0113] The antibody compositions of the invention can be
administered using conventional modes of administration including,
but not limited to, intravenous, intraarterial, intraperitoneal,
oral, intralymphatic, intramuscular, intradermal, subcutaneous, and
intranasal administration. In particular embodiments, the route of
administration is via bolus or continuous infusion over a period of
time, such as continuous or bolus infusion, once or twice a week.
In other particular embodiments, the route of administration is by
subcutaneous injection. The dosage depends on the nature, form, and
stage of the targeted B cell malignancy or autoimmune disease, the
patient's sex, age, condition, prior treatment history, other
treatments used, and other factors typically considered by a
skilled physician. For example, non-Hodgkin's lymphoma patients or
patients with autoimmune disease may receive from about 50 to about
1500 Mg/m.sup.2/week, specifically from about 100 to about 1000
mg/m.sup.2/week, more specifically from about 150 to about 500
mg/m.sup.2/week of an anti-CD22 antibody as described herein.
[0114] The patients can be monitored by standard techniques known
in the art to follow clinical indicia of B-cell malignancy or the
particular autoimmune disease. For example, in the case of B-cell
malignancy, tumor regression (e.g. tumor size in the case of solid
tumors), the phenotype of circulating B-cells or of biopsied
tissues using anti-CD22 antibodies can be monitored.
[0115] While the invention has been discussed with reference to
human therapy, it will be understood that the antibodies of the
present invention also find use in veterinary medicine. For
example, feline malignant lymphoma occurs frequently in domestic
cats, and shows similar characteristics to human non-Hodgkin's
lymphoma (Bertone et al., Am. J. Epidemiol. 156:268-73 (2002)).
Similarly, dogs are known to develop a variety of lymphomas.
Accordingly, the antibodies herein can be used to treat feline and
canine malignant lymphoma. Animal models of autoimmune disease are
also known in the art. Dosages, and routes of administration depend
on the animal species to be treated, and their determination is
well within the skill of a veterinary of ordinary skill.
[0116] Further details of the invention are provided in the
following non-limiting examples.
EXAMPLES
[0117] Commercially available reagents referred to in the examples
were used according to manufacturer's instructions unless otherwise
indicated. In addition to production as disclosed in the following
examples, hybridoma producing monoclonal antibody HB22-7 (ATCC
Accession No. HB11349) may be obtained from the American Type
Culture Collection, Rockville, Md.
EXAMPLE 1
Production of Anti-CD22 Monoclonal Antibodies
[0118] Monoclonal antibodies (mAbs) HB22-7 (IgG2b), HB22-23 (IgG2a)
HB22-33 (IgM), HB22-5 (IgG2a), HB22-13 (IgG2a), HB22-22 (IgA), and
HB22-196 were produced according to the method of Engel et al., J.
Immunol 15:4710 (1993) and U.S. Pat. No. 5,484,892. See, also
Tuscano et al., Blood 94:1382-1392 (1999). However, other methods
may be used. Briefly, the HB22 mAbs were produced via hybridoma
techniques using a mouse pre-B cell line 300.19, stably transfected
with full length CD22 cDNA, as the immunogen. More specifically,
thirty-three mAbs reactive with CD22 were generated by the fusion
of NS-1 myeloma cell with spleen cells from Balb/c mice immunized
three times with a mouse pre-B cell line, 300.19, stably
transfected with a full-length CD22 cDNA. Hybridomas producing mAb
reactive with mouse L cells transfected with CD22 cDNA, but not
with untransfected cells, were cloned twice and used to generate
supernatant or ascites fluid. mAb isotypes were determined using
the Mouse Monoclonal Antibody Isotyping Kit (Amersham, Arlington
Heights, Ill.). IgGmAb were purified using the Affi-Gel Protein A
MAPS II Kit (Bio-Rad, Richmond, Calif.). The HB22-33 mAb (IgM)
containing euglobulin fraction of ascites fluid was precipitated by
extensive dialysis against distilled water and was shown to be
essentially pure mAb by SDS-PAGE analysis. As disclosed in Table II
of U.S. Pat. No. 5,484,892, mAbs HB22-7, HB22-22, HB22-23, and
HB22-33 completely blocked (80-100%) the binding of Daudi, Raji and
Jurkat cells to CD22 transfected COS cells. mAbs HB22-5, HB22-13,
HB22-24, and HB22-28 partially blocked adhesion (20-80%).
[0119] The region(s) on CD22 that mediates ligand binding was
characterized by mAb cross-inhibition studies using the "Workshop"
CD22-blocking mAb and a panel of mAb that identify five different
epitopes on CD22 (epitopes A, B, C, D, and E (Schwartz-Albiez et
al., "The carbohydrate moiety of the CD22 antigen can be modulated
by inhibitors of the glycosylation pathway." The binding
specificities of the Workshop mAb are depicted pictorially in FIG.
3. In Leukocyte Typing IV. White Cell Differentiation Antigens,
Knapp et al., eds., Oxford University Press, Oxford, p. 65 (1989)).
It has been found that three of the monoclonal antibodies herein,
HB22-7, HB22-22, and HB22-23, bind to very close or the same
epitopes on CD22. Results of the epitope-mapping of these and other
blocking antibodies are disclosed in Tedder et al., Annu. Rev.
Immunol. 15:481-504 (1997). Unlike other anti-CD22 antibodies
proposed for therapy, the blocking antibodies of the present
invention bind to an epitope within the first two Ig-like domains
of the hCD22 amino acid sequence.
EXAMPLE 2
Raji and Ramos Lymphoma Xenograft Trials
[0120] This example describes the results from our independent Raji
and Ramos lymphoma xenograft trials. Nude mice xenografts are
important tools for preclinical evaluations. Nude mice bearing
human non-Hodgkin's lymphoma (NHL) xenografts utilizing the
lymphoma cell lines Raji and Ramos have proven utility for
evaluating efficacy for treatment of NHL . (Buchsbaum et al.,
Cancer Res. 52(23):6476-6481 (1992) and Flavell et al., Cancer Res.
57:4824-4829 (1997)).
[0121] Materials and Methods
[0122] Reagents. Carrier-free .sup.90Y (Pacific Northwest National
Laboratory, Richland, Wash.) and .sup.111In (Nordion, Kanata,
Ontario, Canada) were purchased as chlorides in dilute HCl. Lym-1
(Techniclone, Inc Tustin, Calif.) is an IgG.sub.2a mAb generated in
mice immunized with human Burkitt's lymphoma cell nuclei. Lym-1
recognizes a cell surface 31-35 kD antigen on malignant B cells,
and reacts with greater than 80% of human B cell NHL. Lym-1 purity
was assessed according to the specifications that required greater
than 95% pure monomeric IgG by polyacrylamide gel electrophoresis.
.sup.90Y-DOTA-peptide-Lym-1 was prepared as previously described
(O'Donnell et al., Cancer. Biother. Radiopharm. 13:251-361 (1998)).
Assessment by HPLC, TLC, and cellulose acetate electrophoresis
revealed that .sup.90Y-DOTA-peptide-Lym-1 was prepared to 98%
radiochemical purity with less than 5% aggregate content.
[0123] The anti-CD22 mAb, HB22-7, was prepared as previously
described (Tuscano et al., Blood 94:1382-1392 (1999)), using a
Protein A Sepharose Fast Flow column (Pharmacia). HB22-7 purity was
determined by HPLC and flow cytometry, and found to be >95%
pure. Physiologic properties were determined by flow
cytometric-based analysis of apoptotic induction (Apo-Tag,
Pharmacia) and found to be consistent with previous published
results (Tuscano et al., supra). Endotoxin removal was achieved
using an ActiClean ETOX column (Sterogene), with final endotoxin
levels determined to be <0.15 Endotoxin Units (EU)/mg mAb (Bio
Whitaker). The Lym-1 and HB22-7 mAbs met MAP (mouse antibody
production) guidelines for murine, viral, mycoplasma, fungal, and
bacterial contamination, as well as endotoxin, pyrogen and DNA
content and general safety testing in animals.
[0124] Cell lines and Scatchard Analysis. Raji and Ramos Burkitt
lymphoma cell lines were purchased from American Type Culture
Collection (ATCC, Gathersberg, Md.). Both cell lines stained for
CD22 expression by flow cytometric methods utilizing the HB22-7
mAb, as described previously (Tuscano et al., supra). The cell
lines were maintained in RPMI 1640 supplemented with 10% fetal calf
serum at 0.5.times.10.sup.6 cells/ml. A Scatchard analysis using
Raji and Ramos cells was performed as described previously
(Scatchard, G., Ann. of NY Acad Sci. 51:660 (1947)). Briefly,
HB22-7 was labeled with .sup.125I by the chloramine T method
(specific activity of 1.1 .mu.Ci/.mu.g). A competitive binding
assay was performed utilizing serially diluted, unlabeled
HB22-7.
[0125] Mouse studies. Female athymic BALB/c nu/nu mice (Harlan
Sprague-Dawley), 7-9 weeks of age were maintained according to
University of California, Davis animal care guidelines on a normal
diet ad libitum and under pathogen-free conditions. Five mice were
housed per cage. Raji or Ramos cells were harvested in logarithmic
growth phase; 2.5-5.0.times.10.sup.6 cells were injected
subcutaneously into both sides of the abdomen of each mouse.
Studies were initiated 3 weeks after implantation, when tumors were
28-328 mm.sup.3. Groups consisted of untreated, 125 .mu.Ci of RIT
alone, 1.4 mg of HB22-7 alone, or the combination of RIT and
HB22-7, with HB22-7 being administered 24 hours prior,
simultaneously, or 24 hours after RIT. To minimize ambient
radiation, bedding was changed daily for 1 week after treatment
with .sup.90Y-DOTA-peptide-Lym-1, and twice weekly thereafter.
[0126] Tumoricidal Effect. Tumor volume was calculated as described
by the formula for hemiellipsoids (DeNardo et al., Clin. Cancer
Res. 3:71-79 (1997)). Initial tumor volume was defined as the
volume on the day prior to treatment. Mean tumor volume was
calculated for each group on each day of measurement; tumors that
had completely regressed were considered to have a volume of zero.
Tumor responses were categorized as follows: C, cure (tumor
disappeared and did not regrow by the end of the 84 day study); CR,
complete regression (tumor disappeared for at least 7 days, but
later regrew); PR, partial regression (tumor volume decreased by
50% or more for at least 7 days, then regrew).
[0127] Statistical Analysis. Differences in response among
treatment groups were evaluated using the Kruskall Walis rank sum
test with the response ordered as none, PR, CR, and Cure. Survival
time was also evaluated using the Kruskall Walis test. Tumor volume
was compared at 3 time points: month 1 (day 26-29), month 2 (day
54-57), and at the end of the study (day 84). If an animal was
sacrificed due to tumor-related causes, the last volume was carried
forward and used in the analysis of later time points. Analysis of
variance was used to test for differences among treatment groups. P
values are two-tailed and represent the nominal p-values.
Protection for multiple comparisons is provided by testing only
within subsets of groups found to be statistically significantly
different.
[0128] Results
[0129] Scatchard Analysis
[0130] Scatchard analysis was utilized to assess the binding
affinity of HB22-7 and the number of CD22 receptors on Ramos and
Raji cells. The cells were assayed for maximum binding percentage
(Bmax), disassociation constant (Ka) and number of antibodies bound
per cell. The results shown in Table 1 are the average of two
experiments.
1TABLE 1 I. PARAMETER Cell Lines Cell line Raji Ramos Bmax 53.5
.+-. 0.9% 21.0 = 1.3% R.sup.2 0.954 0.926 Ka 1.3 .+-. 0.08 .times.
10.sup.9 5.95 .+-. 1.0 .times. 10.sup.8 Antibody/cell 118,000
43,000
[0131] The Scatchard analysis (Table 1) revealed a nearly 2.5 fold
increase in the number of HB22-7 antibodies bound per cell, and
Bmax, and a 2 fold increase in Ka for Raji cells versus Ramos
cells, respectively.
[0132] Whole Body Autoradiography
[0133] In order to assess HB22-7-specific tumor targeting, whole
body autoradiography of tumor-bearing nude mice injected with
.sup.111In-2IT-BAD-anti-CD22 (HB22-7) was performed. Forty eight
hours after injection mice were sacrificed, sectioned and
autoradiographed (FIG. 2), as previously described (DeNardo et al.,
Cancer 3:71-79 (1997)). Autoradiography revealed intense tumor
localization in the Raji-tumored mice and moderate localization in
the Ramos-tumored mice. This targeting study is consistent with the
Scatchard analysis that revealed less HB22-7 bound per Ramos cells
as compared to Raji. However the rapid growth of Ramos tumors, and
likely central necrosis, may also contribute to the apparent
inferior targeting of Ramos.
[0134] Efficacy of RIT and CMRIT
[0135] The initial trial (081500) utilized 125 uCi of
.sup.90Y-DOTA-peptide-Lym-1 alone or in combination with HB22-7
(1.4 mg) given either 24 hours prior, simultaneously, or 24 hours
after RIT, (FIG. 3). In this trial there were 5 mice per group with
the exception of the group treated with RIT alone, which had 9 mice
and 5 untreated controls (mouse numbers are tabulated in Table
2).
2 TABLE 2 Treatment Groups Trial No Tx HB22-7 RIT -24 @RIT +24
081500 5 4 9 5 5 5 101600 5 6 5 5 3 5 011601 -- 5 4 -- 9 7 032701
-- 5 2 -- 3 12 052401 3 -- 3 -- -- -- 060401 5 5 -- -- -- -- 071701
7 5 -- -- -- 4 092101 4 -- -- -- -- -- 102401 13 -- -- -- -- --
Total 42 30 23 10 20 33
[0136] As predicted from similar Raji xenograft studies with
.sup.90Y-2IT-BAD-Lym-1, RIT alone resulted in maximal mean tumor
volume reduction by day 21, with increasing tumor volume
thereafter. Xenografts treated with .sup.90Y-2IT-BAD-Lym-1(RIT) and
HB22-7 (CMRIT) demonstrated greater and more sustained mean tumor
volume reduction, which was greatest when HB22-7 was administered
simultaneously, and 24 hours after RIT. Surprisingly, HB22-7
administered alone resulted in stabilization of mean tumor volume
by 2-3 weeks, then a gradual and sustained tumor volume
reduction.
[0137] Several additional replicate trials were conducted with
highly reproducible results (Table 2). The data from all trials
were compiled and, when compared graphically, revealed results
highly consistent with the initial study, (FIG. 4). The initial
tumor volume reductions were again greatest at approximately day 21
when HB22-7 was administered simultaneously and 24 hours after RIT.
In mice treated with HB22-7 alone, the stabilization in tumor
growth that began 2 weeks after treatment followed by gradual
sustained tumor volume reduction was also replicated in all
subsequent trials. Using analysis of variance, when examining all
treatment groups at day 30 the differences were highly significant
(p<0.001). While analysis of volume reduction in all treatment
groups at day 60 did not demonstrate significant differences
(p=0.39), the differences at day 84 again were significant
(p=0.003). The results observed graphically revealed that the
difference in volume reduction in the RIT/CMRIT groups was highly
reproducible and different from HB22-7 alone and untreated control,
however, comparison of volume reduction only in only RIT treatment
groups (including CMRIT) at all time points assessed (day 30, 60,
and 84) did not reveal significant differences (p.gtoreq.0.5).
Additional CMRIT trials were done with HB22-7 being administered 48
and 72 hours after RIT. The extended interval between the
administration of RIT and HB22-7 did not result in improved tumor
volume reduction when compared to trials in which HB22-7 was given
simultaneously and 24 hours after RIT (data not shown).
[0138] Response and cure rates were consistent with the effects of
treatment on tumor volume, (FIG. 5). Treatment with
.sup.90Y-DOTA-peptide-Lym-1 alone produced 48% PR, 13% CR, and a
13% cure rate. In the CMRIT groups, the overall response rate was
maximized when HB22-7 and RIT were administered simultaneously
generating 45% PR, 15% CR and 25% cure. However in the CMRIT groups
the cure rate was the greatest (39%) when HB22-7 was administered
24 hours after RIT, which compared favorably to the cure rates
observed in the untreated (29%), RIT alone (13%), 24 hours prior
(10%) and simultaneous (25%) treatment groups. When examining the
degree of response (ranking cure better than CR, better than PR) in
all treatment groups using the Kruskal Walis test, the differences
were statistically significant (p=0.01). Individual comparisons
against untreated controls were all statistically significant
(p<0.05), with the exception of RIT alone (p=0.06) and HB22-7
given 24 hours prior to RIT (p=0. 16). While comparison of only
active treatment groups (RIT alone, CMRIT, and HB22-7) was not
significantly different (p=0.18), the CMRIT groups treated with
HB22-7 simultaneously and after 24 hours had the best observed
pattern of response. Interestingly the group treated with HB22-7
alone had the highest cure rate (47%) which was a significant
improvement when compared to the untreated controls
(p<0.05).
[0139] Tumor volume regression and cure rates translated into a
similar pattern of survival. At the end of the 84 day study period
38 and 42% of the untreated and RIT alone groups were alive
respectively, (FIG. 6). In the CMRIT treatment groups, survival
increased to 67 and 50% when HB22-7 was administered simultaneously
and 24 hours after RIT, respectively. Analysis of survival using
Kruskal Walis was significant (p<0.05) for comparison of all
groups. Similar to the response rate analysis, comparison of
survival in the RIT groups only did not reveal significant
differences (p=0.41), however the best survival in these groups was
consistently observed when HB22-7 was administered either
simultaneous or 24 hours after RIT.
[0140] The best overall survival, 76%, was observed in the group
treated with HB22-7 alone, a significant difference when compared
to untreated control (p=0.02).
[0141] Toxicity
[0142] Hematologic and non-hematologic toxicities were assessed by
blood counts and mouse weights, respectively (FIG. 7a-c). WBC and
platelet nadirs in the RIT treatment groups were at 14-20, and
10-14 days respectively. WBC and platelet recovery was
approximately 28 and 21 days after treatment, respectively. The WBC
and platelet nadirs were consistent with observations in previous
studies that utilized 150 uCi of 90Y-2IT-BAD-Lym-1. The hematologic
toxicity of RIT was not altered by co-administration of HB22-7. No
hematologic toxicity was detected in mice treated with HB22-7
alone. Analysis of mononuclear cell counts in all treatment groups
revealed that HB22-7 had no effect on RIT-mediated mononuclear cell
nadirs (data not shown). Non-hematologic toxicity as assessed by
changes in mouse weight, and was found to be equivalent in all
treatment groups (FIG. 8). There were no deaths due to toxicity in
any treatment groups.
[0143] .sup.90Y-DOTA-Peptide-Lym-1 Pharmacokinetics
[0144] Blood and whole body clearances of 90Y-DOTA-peptide-Lym-1 in
Raji-tumored mice with or without HB22-7 were similar (FIG. 9). The
blood biological T.sub.1/2 a was 1.4 hours for RIT alone, and 2.2,
2.4, and 2.0 hours for the 24 hour prior, simultaneous and 24 hour
after groups respectively. The blood biological T.sub.1/2 .beta.
was 127 hours for the RIT alone group and 133, 87, and 103 hours
for the 24 hours prior, simultaneous and 24 hours after groups
respectively. The whole body T.sub.1/2 was 246 hours for RIT alone
and 207, 207, and 196 hours for the 24 hours prior, simultaneous
and 24 hours after groups respectively. The addition of HB22-7 to
RIT did not change the pharmacokinetics of
.sup.90Y-DOTA-peptide-Lym-1.
Discussion
[0145] Raji xenograft studies were designed to determine if the
anti-CD22 mAb (HB22-7) would generate additive or synergistic
effects when combined with RIT to enhance apoptosis and/or DNA
damage induced by low dose-rate radiation. The Raji xenograft nude
mouse model has proven useful when used to assess toxicity and
efficacy of RIT using .sup.90Y-2IT-BAD-Lym-1 RIT alone (O'Donnell
et al., Cancer Biotherapy and Radiopharmaceuticals 13:351-361
(1998)). Responses in this pre-clinical model translated into
significant efficacy in human clinical trials (O'Donnell et al.,
Anticancer Res. 20:3647-55 (2000); O'Donnell et al, J. Nucl. Med.
40:216 (1999) (Abstract)).
[0146] In the studies described in this Example, the addition of
the anti-CD22 mAb HB22-7 to .sup.90Y-DOTA-peptide-Lym-1(125 uCi)
enhanced the efficacy of RIT without any change in toxicity.
Previous Raji xenograft studies with 150 and 200 uCi of
.sup.90Y-2IT-BAD-Lym-1 generated response and cure rates that were
comparable to those observed in the present study (O'Donnel et al.,
(1998), supra). The 125 .mu.Ci dose of .sup.90Y-DOTA-peptide-Lym-1
was chosen based on these previous studies with the 2IT-BAD linker.
While the previous studies with 2IT-BAD demonstrated greatest
efficacy with the 200 .mu.Ci dose, the choice of 125 .mu.Ci was
based on the hypothesis that HB22-7 would be synergistic or
additive with RIT and the lower dose would allow for better
assessment of these effects. The studies of this Example utilized a
novel linker (DOTA-peptide) that has not been previously examined
in lymphoma xenograft models. The DOTA-peptide linker was designed
for enhanced hepatic degradation of unbound radiopharmaceutical
thereby leading to a more favorable biodistribution. While
tumor-specific uptake was not assessed in detail in this study, the
toxicity profile observed with 125 uCi of
.sup.90Y-DOTA-peptide-Lym-1 alone was acceptable with no
treatment-related mortality and predictable leukocyte and platelet
nadirs.
[0147] HB22-7 was chosen based on in vitro studies demonstrating
pro-apoptotic and signaling effects (Tuscano et al., Blood
94:1382-1392 (1999)). The treatment dose of HB22-7 utilized was
empiric, however, it was based on the amount that was shown to be
effective at inducing apoptosis in vitro and extrapolating this to
the mouse model. In addition, when formulating the dose of HB22-7
consideration was given to the equivalent (when adjusted for body
surface area differences in humans versus mice) dose of
Rituximab.RTM. used in human clinical trials. The approximation to
the Rituximab.RTM. dose was utilized based on the fact that this is
the only naked mAb available that has demonstrated efficacy for the
treatment of lymphoma, granted, the optimal dose of Rituximabo is
currently undefined.
[0148] The study was designed to assess the efficacy of HB22-7
alone, the combination of RIT and HB22-7 as well as the effect of
three different sequence combinations. The tumor volume reduction
observed with .sup.90Y-DOTA-peptide-Lym-1 alone was consistent with
previous studies with .sup.90Y-2IT-BAD-Lym-1 in terms of timing,
magnitude, and duration of response (O'Donnel et al., 1998, supra).
RIT alone resulted in approximately 50% reduction in tumor volume
14 days after therapy. When assessing at the approximate point of
maximal volume reduction (day 21-30) the addition of HB22-7 to RIT
significantly enhanced the magnitude of response in a sequence
specific manner. It appears that the addition of HB22-7 was most
effective when administered simultaneously or 24 hours after RIT.
The distinctive pattern of volume reduction was highly
reproducible. Independent replicate trials demonstrated similar
patterns and magnitude of tumor volume reduction. The improved
reductions in tumor volume translated into superior response rates
and survival. RIT alone generated 13% CR and 13% cures, the
addition of HB22-7 increased the cure rate to 25% when administered
simultaneously with RIT, and to 39% when HB22-7 was administered 24
hours after RIT.
[0149] This is the first time that a second monoclonal antibody has
been combined with RIT, and demonstrates the potential of utilizing
monoclonal antibodies or other agents with well defined physiologic
properties that may augment efficacy without increasing
toxicity.
[0150] Surprisingly the mice treated with HB22-7 alone had
impressive tumor volume reduction and superior cure and survival
rates when compared to all other treatment groups. Again, several
independent trials generated highly consistent results with a
delayed initial tumor volume stabilization, and then tumor volume
reduction beginning approximately 14 days after treatment. This
translated into the best cure and overall survival rates observed
in any of the treatment groups.
[0151] In conclusion, the antibodies of the present invention, when
administered alone, have been demonstrated to provide superior
results in terms of tumor volume reduction, cure rate and overall
survival when compared to other treatment regimens, including
CMRIT, which is currently viewed as the most advanced therapeutic
approach for the treatment of NHL.
EXAMPLE 3
Sequence Analysis of Anti-CD22 Antibodies
[0152] V.sub.H and Light Chain Gene Utilization
[0153] Cytoplasmic RNA was extracted from 1-10.times.10.sup.5
hybridoma cells using the RNeasy Mini Kit (Qiagen Chatsworth,
Calif.). First strand cDNA was synthesized from cytoplasmic RNA
using oligo-dT primers (dT.sub.18) and a Superscript Kit (Gibco
BRL, Gaithersburg, Md.). One .mu.l of cDNA solution was used as
template for PCR amplification of V.sub.H genes. PCR reactions were
carried out in a 100-.mu.l volume of a reaction mixture composed of
10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl.sub.2, 200 .mu.M
dNTP (Perkin Elmer, Foster City, Calif.), 50 pmol of each primer,
and 5 U of Taq polymerase (ISC Bioexpress, Kaysville, Utah).
Amplification was for 30 cycles (94.degree. C. for 1 min,
58.degree. for 1 min, 72.degree. C. for 1 min; Thermocycler, Perkin
Elmer). V.sub.H genes were amplified using a promiscuous sense 5'
V.sub.H primer (Ms V.sub.HE: 5' GGG AAT TCG AGG TGC AGC TGC AGG AGT
CTG G 3'; SEQ ID NO: 2) as previously described (Kantor et al., J.
Immunol. 158:1175-86 (1996)), and antisense primers complementary
to the C.mu. coding region (primer C.mu.-in: 5' GAG GGG GAC ATT TGG
GAA GGA CTG 3'; SEQ ID NO: 3) or the C.gamma. region (Primer
C.gamma.1: 5' GAG TTC CAG GTC ACT GTC ACT GGC 3'; SEQ ID NO:
4).
[0154] Light chain cDNA was amplified using a sense V.kappa. primer
[5' ATG GGC (AT)TC AAG ATG GAG TCA CA(GT) (AT)(CT)(CT) C(AT)G G 3';
SEQ ID NO: 5] and a C.lambda. antisense primer (5' ACT GGA TGG TGG
GAA GAT G 3'; SEQ ID NO: 6).
[0155] HB22-33 light chain sequences were amplified using a
different sense V.kappa. primer (5' ATG AAG TTG CCT GTT AGG CTG TTG
GTG CTG 3'; SEQ ID NO: 7).
[0156] Amplified PCR products were purified from agarose gels using
the QIAquick gel purification kit (Qiagen) and were sequenced
directly in both directions using an ABI 377 PRISM DNA sequencer
after amplification using the Perkin Elmer Dye Terminator
Sequencing system with AmpliTaq DNA polymerase and the same primers
for initial PCR amplification. All V.sub.H and light chain regions
were sequenced completely on both the sense and anti-sense DNA
strands.
[0157] The alignment of the V.sub.H and V.kappa. amino acid
sequences for anti-CD22 monoclonal antibodies HB22-5, HB22-7,
HB22-13, HB22-23, HB22-33, and HB22-196 are shown in FIGS. 10 and
17, respectively. FIGS. 11-16 show the nucleotide and amino acid
sequences for heavy chain V.sub.H-D-J.sub.H junctions of anti-CD22
Abs from hybridomas HB22-5 (SEQ ID NOS: 8 and 9), HB22-7 (SEQ ID
NOS: 10 and 11); HB-22-13 (SEQ ID NOS: 12 and 13); HB-22-23 (SEQ ID
NOS: 14 and 15); HB-22-33 (SEQ ID NOS: 16 and 17); and HB-22-196
(SEQ ID NOS: 18 and 19). FIGS. 18-23 show the nucleotide and
deduced amino acid sequences for kappa light chain V-J-constant
region junctions of anti-CD22 Abs from hybridomas HB22-5 (SEQ ID
NOS: 20 and 21); HB22-7 (SEQ ID NOS: 22 and 23); HB22-13 (SEQ ID
NOS: 24 and 25) HB22-23 (SEQ ID NOS: 26 and 27); HB22-33 (SEQ ID
NOS: 28 and 29); and HB22-196 (SEQ ID NOS: 30 and 31).
* * * * *