Therapeutic Sirp-alpha Antibodies

Abstract

Anti-SIRP.alpha. monoclonal antibodies (anti-SIRP.alpha. mAbs), including multispecific SIRP.alpha. antibodies, are provided with distinct functional profiles as are related compositions and methods of using anti-SIRP.alpha. mAbs as therapeutics for the prevention and treatment of solid and hematological cancers. Also provided are amino acid sequences of exemplary anti-SIRP.alpha. monoclonal antibodies.

Description

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation of U.S. patent application Ser. No. 16/682,893, filed Nov. 13, 2019, which claims priority to U.S. Provisional Application No. 62/931,746, filed Nov. 6, 2019, U.S. Provisional Application No. 62/886,872, filed Aug. 14, 2019, U.S. Provisional Application No. 62/820,718, filed Mar. 19, 2019, and U.S. Provisional Application No. 62/767,509, filed Nov. 14, 2018, each of which is herein incorporated by reference in their entireties for all purposes.

SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Nov. 13, 2021, is named ARCO_010_08US_SeqList.txt and is 146 kilobytes in size.

FIELD OF THE DISCLOSURE

[0003] This disclosure pertains to the field of immunotherapy. The present disclosure provides anti-SIRP.alpha. antibodies (anti-SIRP.alpha.) which disrupt the interaction between SIRP.alpha. and CD47, enhance phagocytosis of tumor cells, cause immunomodulation of immune responses, and methods to generate anti-SIRP.alpha. antibodies and use anti-SIRP.alpha. antibodies as therapeutic agents for the prevention and treatment of hematological and solid and cancers.

BACKGROUND

[0004] Therapeutic antibodies targeting adaptive immunity including the T-cell checkpoints, PD-1, PD-L1 and CTLA-4 to enhance the cytotoxic activity of the T-cell immune response have raised the prospect of long-term remission or even cure for patients with metastatic diseases (Hodi 2010, McDermott 2015). Despite positive results, there remains a significant patient population that either fails to respond to these checkpoint inhibitors (primary resistance) or those that respond, but eventually develop disease progression (acquired resistance) (Pitt 2016, Restifo 2016, Sharma 2017). Recent studies suggest that resistance mechanisms can be both tumor cell intrinsic, including a lack of unique tumor antigen proteins or inhibition of tumor antigen presentation, and tumor cell extrinsic, involving the absence of infiltrating T-cells, redundant inhibitory checkpoints and/or the presence of immunosuppressive cells in the tumor microenvironment (Sharma 2017). Even in tumors considered sensitive to checkpoint inhibitors, or when combining anti-CTLA-4 and anti-PD-1/PDL-1 agents, approximately 50% of patients do not experience tumor shrinkage and the median treatment duration or progression-free survival for all treated patients remains relatively short around 2-5 months (Kazandjian, 2016). In addition, several of the most prevalent solid tumors and the majority of hematological malignancies have shown disappointing results with these checkpoint inhibitors. In particular, hormone receptor-positive breast cancer, colorectal cancer (non-microsatellite instability) and prostate cancer do not appear to be sensitive to this type of immune manipulation and could benefit from a different immunotherapy approach (Le 2015, Dirix 2015, Topalian 2012, Graff 2016). These findings highlight the need for alternative or synergistic approaches that target additional checkpoints to activate the innate immune response in addition to the adaptive immune response to further improve clinical outcomes. Several checkpoints of the innate immune response are present on tumor cells and on myeloid cells (macrophages, dendritic cells, monocyte-derived suppressor cells, granulocytes) which are important cellular components of the tumor microenvironment that influence tumor progression, metastasis and overall outcome (Barclay and van den Berg 2014, Yanagita 2017).

[0005] SIgnal Regulatory Protein (SIRP)-.alpha. or SIRP.alpha., also known as CD172a, BIT or SHPS-1, is a member of the SIRP paired receptor family of closely related SIRP proteins. SIRP.alpha. is expressed mainly by hematopoietic cells, including macrophages, dendritic cells and granulocytes, and is also expressed on neurons, especially in the brain, glia, smooth muscle cells and endothelial and some tumor cells (Barclay and van den Berg 2014). SIPR.alpha. is a transmembrane protein with an extracellular domain containing three Ig-like domains and a cytoplasmic region that contains immunoreceptor tyrosine-based inhibitory motifs (ITIMs). The gene encoding human SIRP.alpha. is polymorphic with two common variants identified, SIRP.alpha.V1 and SIRP.alpha.V2, with changes in surface amino acids, but that do not appear to affect binding to its ligand, cluster of differentiation 47 (CD47) (Barclay and van den Berg 2014). The interaction of SIRP.alpha., expressed by myeloid cells, with CD47, expressed or overexpressed on many tumor cells as well as on normal cells, is an important immune checkpoint of the innate response that regulates myeloid functions that include adhesion, migration, activation and inhibitory activities. The CD47/SIRP.alpha. interaction regulates macrophage and dendritic cell phagocytosis of target cells sending an inhibitory "don't eat me signal" to the phagocyte. The binding of CD47 to SIRP.alpha. initiates an inhibitory signaling cascade resulting in inhibition of phagocytosis following phosphorylation of its cytoplasmic ITIMs (Oldenborg 2000, Oldenborg 2001, Okazawa 2005), recruitment and binding of SHP-1 and SHP-2, Src homology domain-containing protein tyrosine phosphatases (Veillette 1998, Oldenborg 2001), inhibition of non-muscle myosin IIA and ultimately phagocytic function (Tsai and Discher 2008, Barclay and van den Berg 2014, Murata 2014, Veillette and Chen 2018, Matazaki 2009). An important corollary of the action of CD47 as a "don't eat me" signal is its role as a "marker of self". This provides a significant hindrance to phagocytosis of self and blocks a subsequent autoimmune response (Oldenborg, 2002, Oldenborg 2004). Cancer cells use CD47 to mask themselves in "selfness" consequently evading both the innate and adaptive immune systems. Blocking the interaction SIRP.alpha. on innate immune cells such as macrophages and dendritic cells with CD47 on tumor cells has emerged as a viable target in cancer therapy. Preclinical data has indicated that, similar to anti-CD47 antibodies, anti-SIRP.alpha. antibodies that block the SIRP.alpha./CD47 interaction exhibit anti-tumor efficacy in mouse tumor models, either as monotherapy or in combination with other agents (Gauttier, 2017; Ring, 2017; Yanigita, 2017; Poirier, 2018; and Guattier, 2018). Importantly, generation of an adaptive immune response, in addition to the innate immune response following interruption of the SIRP.alpha./CD47 interaction, appears to be critical to obtaining a robust anti-tumor response (Tseng 2013, Li 2015, Xu 2017).

[0006] Expression of SIRP.alpha. on DC cells and its interaction with CD47 on T-cells appears to be important in inducing the adaptive immune response. Blockade of the SIRP.alpha./CD47 interaction was reported to affect the DCs ability to stimulate the antigen-specific CD8+ T-cell response and this was correlated with an enhanced DC-mediated response to tumor DNA (Liu 2015, Xu 2017).

[0007] Another member of the SIRP family of paired receptors, SIRP-.gamma., is selectively expressed on the surface of human (but not rodent) T-cells, has a short cytoplasmic region consisting of 4 amino acids. SIRP-.gamma. also binds to CD47 and appears to be important for mediating adhesion between T-cell and APC and for T-cell functions including proliferation and activation (Barclay and van den Berg 2014; and Piccio, 2005). Thus, blocking the interaction between SIRP.alpha. and CD47 but not between SIRP-.gamma. and CD47 may provide an advantage to protecting T-cell function.

[0008] The present disclosure describes anti-SIRP.alpha. mAbs with distinct functional profiles. The antibodies of the disclosure are useful in various therapeutic methods for treating diseases and conditions associated with SIRP.alpha. in humans, including using anti-SIRP.alpha. mAbs as therapeutics for the prevention and treatment of solid and hematological cancers. The antibodies of the disclosure are also useful as diagnostics to determine the level of anti-SIRP.alpha. expression in tissue samples. Embodiments of the disclosure include isolated antibodies and immunologically active binding fragments thereof; pharmaceutical compositions comprising one or more of the anti-SIRP.alpha. monoclonal antibodies, preferably chimeric or humanized forms of said antibodies; and methods of therapeutic use of such anti-SIRP.alpha. monoclonal antibodies.

[0009] The embodiments of the disclosure include the mAbs, or antigen-binding fragments thereof, which are defined by reference to specific structural characteristics, i.e., specified amino acid sequences of either the CDRs or entire heavy and light-chain variable domains or entire heavy- and light-chains. All of these antibodies disclosed herein bind to either SIRP.alpha., SIRP.gamma., or SIRP.alpha. and SIRP.gamma..

[0010] The monoclonal antibodies, or antigen binding fragments thereof may comprise at least one, usually at least three, CDR sequences as provided herein, usually in combination with framework sequences from a human variable region or as an isolated CDR peptide. In some embodiments, an antibody comprises at least one light-chain comprising the three light-chain CDR sequences provided herein situated in a variable region framework, which may be, without limitation, a murine or human variable region framework, and at least one heavy-chain comprising the three heavy-chain CDR sequences provided herein situated in a variable region framework, which may be, without limitation, a human or murine variable region framework.

[0011] In some embodiments the combinations of 6 CDRs include, but are not limited to, the combinations of variable heavy-chain CDR1 (HCDR1), variable heavy-chain CDR2 (HCDR2), variable heavy-chain CDR3 (HCDR3), variable light-chain CDR1 (LCDR1), variable light-chain CDR2 (LCDR2), and variable light-chain CDR3 (LCDR3) selected from:

[0012] HCDR1 comprising SEQ ID NO:33, HCDR2 comprising SEQ ID NO:34, HCDR3 comprising SEQ ID NO:35, LCDR1 comprising SEQ ID NO:1, LCDR2 comprising SEQ ID NO:2, LCDR3 comprising SEQ ID NO:3;

[0013] HCDR1 comprising SEQ ID NO:36, HCDR2 comprising SEQ ID NO:37, HCDR3 comprising SEQ ID NO:38, LCDR1 comprising SEQ ID NO:4, LCDR2 comprising SEQ ID NO:5, LCDR3 comprising SEQ ID NO:6;

[0014] HCDR1 comprising SEQ ID NO:39, HCDR2 comprising SEQ ID NO:40, HCDR3 comprising SEQ ID NO:41, LCDR1 comprising SEQ ID NO:7, LCDR2 comprising SEQ ID NO:8, LCDR3 comprising SEQ ID NO:9;

[0015] HCDR1 comprising SEQ ID NO:42, HCDR2 comprising SEQ ID NO:43, HCDR3 comprising SEQ ID NO:44, LCDR1 comprising SEQ ID NO:10, LCDR2 comprising SEQ ID NO:11, LCDR3 comprising SEQ ID NO:12;

[0016] HCDR1 comprising SEQ ID NO:45, HCDR2 comprising SEQ ID NO:46, HCDR3 comprising SEQ ID NO:47, LCDR1 comprising SEQ ID NO:13, LCDR2 comprising SEQ ID NO:14, LCDR3 comprising SEQ ID NO:15;

[0017] HCDR1 comprising SEQ ID NO:48, HCDR2 comprising SEQ ID NO:49, HCDR3 comprising SEQ ID NO:50, LCDR1 comprising SEQ ID NO:16, LCDR2 comprising SEQ ID NO:17, LCDR3 comprising SEQ ID NO:18;

[0018] HCDR1 comprising SEQ ID NO:51, HCDR2 comprising SEQ ID NO:52, HCDR3 comprising SEQ ID NO:53, LCDR1 comprising SEQ ID NO:19, LCDR2 comprising SEQ ID NO:20, LCDR3 comprising SEQ ID NO:21.

[0019] HCDR1 comprising SEQ ID NO:54, HCDR2 comprising SEQ ID NO:55, HCDR3 comprising SEQ ID NO:56, LCDR1 comprising SEQ ID NO:22, LCDR2 comprising SEQ ID NO:23, LCDR3 comprising SEQ ID NO:24.

[0020] HCDR1 comprising SEQ ID NO:57, HCDR2 comprising SEQ ID NO:58, HCDR3 comprising SEQ ID NO:59, LCDR1 comprising SEQ ID NO:25, LCDR2 comprising SEQ ID NO:26, LCDR3 comprising SEQ ID NO:27.

[0021] HCDR1 comprising SEQ ID NO:60, HCDR2 comprising SEQ ID NO:61, HCDR3 comprising SEQ ID NO:62, LCDR1 comprising SEQ ID NO:28, LCDR2 comprising SEQ ID NO:29, LCDR3 comprising SEQ ID NO:30.

[0022] HCDR1 comprising SEQ ID NO:42, HCDR2 comprising SEQ ID NO:43, HCDR3 comprising SEQ ID NO:44, LCDR1 comprising SEQ ID NO:10, LCDR2 comprising SEQ ID NO:31, LCDR3 comprising SEQ ID NO:12.

[0023] HCDR1 comprising SEQ ID NO:42, HCDR2 comprising SEQ ID NO:43, HCDR3 comprising SEQ ID NO:44, LCDR1 comprising SEQ ID NO:10, LCDR2 comprising SEQ ID NO:31, LCDR3 comprising SEQ ID NO:32.

[0024] HCDR1 comprising SEQ ID NO:57, HCDR2 comprising SEQ ID NO:58, HCDR3 comprising SEQ ID NO:63, LCDR1 comprising SEQ ID NO:25, LCDR2 comprising SEQ ID NO:26, LCDR3 comprising SEQ ID NO:27.

[0025] In some embodiments, the anti-SIRP.alpha. antibodies include antibodies or antigen binding fragments thereof, comprising a heavy-chain variable domain (V.sub.H) having an amino acid sequence selected from the amino acid sequences of: SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, and SEQ ID NO:97, and amino acid sequences exhibiting at least 85%, 90%, 95%, 97%, 98%, or 99% sequence identity to one of the recited sequences. Alternatively or in addition, anti-SIRP.alpha. antibodies, including antibodies or antigen binding fragments thereof, may comprise a light-chain variable domain (V.sub.L) having an amino acid sequence selected from the amino acid sequences of SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, and SEQ ID NO:80, and amino acid sequences exhibiting at least 85%, 90%, 95%, 97%, 98%, or 99% sequence identity to one of the recited sequences.

[0026] Although all possible pairing of V.sub.H domains and V.sub.L domains selected from the V.sub.H domain and V.sub.L domain sequence groups listed above are permissible, certain combinations of V.sub.H and V.sub.L domains are disclosed. Accordingly, anti-SIRP.alpha. antibodies, or antigen binding fragments thereof, are those comprising a combination of a heavy-chain variable domain (V.sub.H) and a light-chain variable domain (V.sub.L), wherein the combination is selected from: [0027] i. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:81 and a light chain variable domain comprising the amino acid sequence SEQ ID NO:64; [0028] ii. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:82 and a light chain variable domain comprising the amino acid sequence SEQ ID NO:65; [0029] iii. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:83 and a light chain variable domain comprising the amino acid sequence SEQ ID NO:66; [0030] iv. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:84 and a light chain variable domain comprising the amino acid sequence SEQ ID NO:67; [0031] v. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:85 and a light chain variable domain comprising the amino acid sequence SEQ ID NO:68; [0032] vi. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:86 and a light chain variable domain comprising the amino acid sequence SEQ ID NO:69; [0033] vii. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:87 and a light chain variable domain comprising the amino acid sequence SEQ ID NO:70; [0034] viii. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:88 and a light chain variable domain comprising the amino acid sequence SEQ ID NO:71; [0035] ix. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:89 and a light chain variable domain comprising the amino acid sequence SEQ ID NO:72; [0036] x. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:90 and a light chain variable domain comprising the amino acid sequence SEQ ID NO:73; [0037] xi. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:91 and a light chain variable domain comprising the amino acid sequence SEQ ID NO:74; [0038] xii. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:91 and a light chain variable domain comprising the amino acid sequence SEQ ID NO:75; [0039] xiii. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:91 and a light chain variable domain comprising the amino acid sequence SEQ ID NO:76; [0040] xiv. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:92 and a light chain variable domain comprising the amino acid sequence SEQ ID NO:74; [0041] xv. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:92 and a light chain variable domain comprising the amino acid sequence SEQ ID NO:75; [0042] xvi. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:92 and a light chain variable domain comprising the amino acid sequence SEQ ID NO:76; [0043] xvii. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:93 and a light chain variable domain comprising the amino acid sequence SEQ ID NO:74; [0044] xviii. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:93 and a light chain variable domain comprising the amino acid sequence SEQ ID NO:75; [0045] xix. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:93 and a light chain variable domain comprising the amino acid sequence SEQ ID NO:76; [0046] xx. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:94 and a light chain variable domain comprising the amino acid sequence SEQ ID NO:74; [0047] xxi. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:94 and a light chain variable domain comprising the amino acid sequence SEQ ID NO:75; [0048] xxii. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:94 and a light chain variable domain comprising the amino acid sequence SEQ ID NO:76; [0049] xxiii. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:84 and a light chain variable domain comprising the amino acid sequence SEQ ID NO:77; [0050] xxiv. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:95 and a light chain variable domain comprising the amino acid sequence SEQ ID NO:78; [0051] xxv. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:95 and a light chain variable domain comprising the amino acid sequence SEQ ID NO:79; [0052] xxvi. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:95 and a light chain variable domain comprising the amino acid sequence SEQ ID NO:80; [0053] xxvii. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:96 and a light chain variable domain comprising the amino acid sequence SEQ ID NO:78; [0054] xxviii. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:96 and a light chain variable domain comprising the amino acid sequence SEQ ID NO:79; [0055] xxix. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:96 and a light chain variable domain comprising the amino acid sequence SEQ ID NO:80; [0056] xxx. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:97 and a light chain variable domain comprising the amino acid sequence SEQ ID NO:78; [0057] xxxi. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:97 and a light chain variable domain comprising the amino acid sequence SEQ ID NO:79; [0058] xxxii. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:97 and a light chain variable domain comprising the amino acid sequence SEQ ID NO:80; and [0059] xxxiii. a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:89 and a light chain variable domain comprising the amino acid sequence SEQ ID NO:72.

[0060] In some embodiments, the anti-SIRP.alpha. antibodies or antigen binding fragments thereof may also comprise a combination of a heavy-chain variable domain and a light-chain variable domain wherein the heavy-chain variable domain comprises a V.sub.H sequence with at least 85% sequence identity, or at least 90% sequence identity, or at least 95% sequence identity, or at least 97%, 98% or 99% sequence identity, to the heavy chain amino acid sequences shown above in (i) to (xxxiii) and/or the light chain variable domain comprises a V.sub.L sequence with at least 85% sequence identity, or at least 90% sequence identity, or at least 95% sequence identity, or at least 97%, 98% or 99% sequence identity, to the light-chain amino acid sequences shown above in (i) to (xxxiii). The specific V.sub.H and V.sub.L pairings or combinations in parts (i) through (xxxiii) may be preserved for anti-SIRP.alpha. antibodies having V.sub.H and V.sub.L domain sequences with a particular percentage sequence identity to these reference sequences.

[0061] For all embodiments the heavy-chain and/or light-chain variable domains of the antibodies or antigen binding fragments are defined by a particular percentage sequence identity to a reference sequence, the V.sub.H and/or V.sub.L domains may retain identical CDR sequences to those present in the reference sequence such that the variation is present only within the framework regions.

[0062] In another embodiment, the anti-SIRP.alpha. antibodies, or antigen binding fragments thereof, are those comprising a combination of a heavy chain (HC) and a light chain (LC), wherein the combination is selected from: [0063] i. a heavy chain comprising the amino acid sequence of SEQ ID NO:109 and a light chain comprising the amino acid sequence SEQ ID NO:98; [0064] ii. a heavy chain comprising the amino acid sequence of SEQ ID NO:110 and a light chain comprising the amino acid sequence SEQ ID NO:99; [0065] iii. a heavy chain comprising the amino acid sequence of SEQ ID NO:111 and a light chain comprising the amino acid sequence SEQ ID NO:100. [0066] iv. a heavy chain comprising the amino acid sequence of SEQ ID NO:112 and a light chain comprising the amino acid sequence SEQ ID NO:101; [0067] v. a heavy chain comprising the amino acid sequence of SEQ ID NO:112 and a light chain comprising the amino acid sequence SEQ ID NO:102; [0068] vi. a heavy chain comprising the amino acid sequence of SEQ ID NO:112 and a light chain comprising the amino acid sequence SEQ ID NO:103; [0069] vii. a heavy chain comprising the amino acid sequence of SEQ ID NO:113 and a light chain comprising the amino acid sequence SEQ ID NO:101; [0070] viii. a heavy chain comprising the amino acid sequence of SEQ ID NO:113 and a light chain comprising the amino acid sequence SEQ ID NO:102; [0071] ix. a heavy chain comprising the amino acid sequence of SEQ ID NO:113 and a light chain comprising the amino acid sequence SEQ ID NO:103; [0072] x. a heavy chain comprising the amino acid sequence of SEQ ID NO:114 and a light chain comprising the amino acid sequence SEQ ID NO:101; [0073] xi. a heavy chain comprising the amino acid sequence of SEQ ID NO:114 and a light chain comprising the amino acid sequence SEQ ID NO:102; [0074] xii. a heavy chain comprising the amino acid sequence of SEQ ID NO:114 and a light chain comprising the amino acid sequence SEQ ID NO:103; [0075] xiii. a heavy chain comprising the amino acid sequence of SEQ ID NO:115 and a light chain comprising the amino acid sequence SEQ ID NO:101; [0076] xiv. a heavy chain comprising the amino acid sequence of SEQ ID NO:115 and a light chain comprising the amino acid sequence SEQ ID NO:102; [0077] xv. a heavy chain comprising the amino acid sequence of SEQ ID NO:115 and a light chain comprising the amino acid sequence SEQ ID NO:103; [0078] xvi. a heavy chain comprising the amino acid sequence of SEQ ID NO:116 and a light chain comprising the amino acid sequence SEQ ID NO:104; [0079] xvii. a heavy chain comprising the amino acid sequence of SEQ ID NO:117 and a light chain comprising the amino acid sequence SEQ ID NO:105; [0080] xviii. a heavy chain comprising the amino acid sequence of SEQ ID NO:117 and a light chain comprising the amino acid sequence SEQ ID NO:106; [0081] xix. a heavy chain comprising the amino acid sequence of SEQ ID NO:117 and a light chain comprising the amino acid sequence SEQ ID NO:107; [0082] xx. a heavy chain comprising the amino acid sequence of SEQ ID NO:118 and a light chain comprising the amino acid sequence SEQ ID NO:105; [0083] xxi. a heavy chain comprising the amino acid sequence of SEQ ID NO:118 and a light chain comprising the amino acid sequence SEQ ID NO:106; [0084] xxii. a heavy chain comprising the amino acid sequence of SEQ ID NO:118 and a light chain comprising the amino acid sequence SEQ ID NO:107; [0085] xxiii. a heavy chain comprising the amino acid sequence of SEQ ID NO:119 and a light chain comprising the amino acid sequence SEQ ID NO:105; [0086] xxiv. a heavy chain comprising the amino acid sequence of SEQ ID NO:119 and a light chain comprising the amino acid sequence SEQ ID NO:106; [0087] xxv. a heavy chain comprising the amino acid sequence of SEQ ID NO:119 and a light chain comprising the amino acid sequence SEQ ID NO:107; and [0088] xxvi. a heavy chain comprising the amino acid sequence of SEQ ID NO:120 and a light chain comprising the amino acid sequence SEQ ID NO:108.

[0089] Various forms of the anti-SIRP.alpha. mAbs are disclosed. For example, the anti-SIRP.alpha. mAbs can be full length humanized antibodies with human frameworks and constant regions of the isotypes, IgA, IgD, IgE, IgG, and IgM, more particularly, IgG1, IgG2, IgG3, IgG4, and in some cases with various mutations to alter Fc receptor function or prevent Fab arm exchange or an antibody fragment, e.g., a F(ab')2 fragment, a F(ab) fragment, a single chain Fv fragment (scFv), etc., as disclosed herein.

[0090] In some embodiments, the anti-SIRP.alpha. mAbs or antigen-binding fragment thereof comprises an IgG isotype selected from IgG1, IgG1-N297Q, IgG2, IgG4, IgG4 S228P, IgG4 PE and variants thereof.

[0091] In some embodiments, the anti-SIRP.alpha. mAbs or antigen-binding fragment thereof binds human SIRP.gamma. in addition to human SIRP.alpha..

[0092] In some embodiments, the anti-SIRP.alpha. mAbs or antigen-binding fragment thereof selectively binds human SIRP.alpha..

[0093] In some embodiments, the anti-SIRP.alpha. mAbs or antigen-binding fragment thereof increases phagocytosis of human tumor cells.

[0094] In some embodiments, the anti-SIRP.alpha. mAbs as disclosed herein are multispecific antibodies that specifically bind to SIRP.alpha. and at least a second antigen, where the second antigen is a marker of a CD47-expressing cell.

[0095] In some embodiments, the second antigen of the multispecific antibody is selected from CD19, CD20, CD22, CD24, CD25, CD30, CD33, CD40, CD44, HER2, CD52, CD56, CD70, CD96, CD97, CD99, CD123, CD279 (PD-1), CD117, C-Met, PTHR2, EGFR, RANKL, SLAMF7, PD-L1, CD38, CD19/CD3, HAVCR2 (TIM3), and GD2.

[0096] In some embodiments, the anti-SIRP.alpha. mAbs or antigen-binding fragment thereof increases phagocytosis of human tumor cells and are administered in combination with an opsonizing monoclonal antibody that targets an antigen on a tumor cell.

[0097] In some embodiments, the anti-SIRP.alpha. mAbs or antigen-binding fragment thereof increases phagocytosis of human tumor cells and are administered in combination with an opsonizing monoclonal antibody that targets an antigen on a tumor cell, wherein the opsonizing monoclonal antibody is chosen from rituximab (anti-CD20), trastuzumab (anti-HER2), alemtuzumab (anti-CD52), cetuximab (anti-EGFR), panitumumab (anti-EGFR), ofatumumab (anti-CD20), denosumab (anti-RANKL), pertuzumab (anti-HER2), panitumumab (EGFR), pertuzumab (HER2), elotuzumab (SLAMF7), atezolizumab (anti-PD-L1), avelumab (anti-PD-L1), durvalumab (anti-PD-L1), necitumumab (anti-EGFR), daratumumab (anti-CD38), obinutuzumab (anti-CD20), blinatumomab (anti-CD19/CD3), dinutuximab (anti-GD2)

[0098] In some embodiments, the opsonizing monoclonal antibody targets CD20, EGFR, and PD-L1.

[0099] In some embodiments, the anti-SIRP.alpha. mAbs or antigen-binding fragment thereof exhibits anti-tumor activity.

[0100] In some embodiments, the anti-SIRP.alpha. mAbs or antigen-binding fragment thereof is administered in combination with an anti-CD47 antibody, wherein the anti-CD47 antibody is described in U.S. Pat. No. 10,239,945, and hereby incorporated by reference in its entirety.

[0101] In some embodiments, the anti-SIRP.alpha. mAbs or antigen-binding fragment thereof is administered in combination with an anti-EGFR antibody.

[0102] In some embodiments, the anti-SIRP.alpha. mAbs or antigen-binding fragment thereof is administered in combination with an anti-PD-1 antibody.

[0103] In some embodiments, the anti-SIRP.alpha. mAbs or antigen-binding fragment thereof is administered in combination with an anti-CTLA-4 antibody.

[0104] In some embodiments, the disclosure provides a pharmaceutical composition comprising one or more of the anti-SIRP.alpha. mAbs or antigen-binding fragments disclosed herein, optionally in chimeric or humanized forms, and a pharmaceutically or physiologically acceptable carrier, diluent, or excipient.

[0105] In some embodiments, the anti-SIRP.alpha. mAbs or antigen-binding fragment thereof are for use in human therapy.

[0106] In some embodiments, the anti-SIRP.alpha. mAbs or antigen-binding fragment thereof are for use in preventing or treating cancer in a human patient.

[0107] Prior to the present disclosure, there was a need to identify anti-SIRP.alpha. mAbs that possess the functional profiles as described herein. The anti-SIRP.alpha. mAbs of the present disclosure exhibit a combination of properties that render the mAbs particularly advantageous for use in human therapy, particularly in the prevention and/or treatment of solid and hematological cancers.

[0108] In some embodiments, the cancer is selected from leukemia, a lymphoma, multiple myeloma, ovarian cancer, breast cancer, endometrial cancer, colon cancer (colorectal cancer), rectal cancer, bladder cancer, urothelial cancer, lung cancer (non-small cell lung cancer, adenocarcinoma of the lung, squamous cell carcinoma of the lung), bronchial cancer, bone cancer, prostate cancer, pancreatic cancer, gastric cancer, hepatocellular carcinoma, gall bladder cancer, bile duct cancer, esophageal cancer, renal cell carcinoma, thyroid cancer, squamous cell carcinoma of the head and neck (head and neck cancer), testicular cancer, cancer of the endocrine gland, cancer of the adrenal gland, cancer of the pituitary gland, cancer of the skin, cancer of soft tissues, cancer of blood vessels, cancer of brain, cancer of nerves, cancer of eyes, cancer of meninges, cancer of oropharynx, cancer of hypopharynx, cancer of cervix, and cancer of uterus, glioblastoma, meduloblastoma, astrocytoma, glioma, meningioma, gastrinoma, neuroblastoma, melanoma, myelodysplastic syndrome, and a sarcoma.

[0109] In some embodiments, the leukemia is selected from leukemia is selected from the group consisting of systemic mastocytosis, acute lymphocytic (lymphoblastic) leukemia (ALL), T-cell--ALL, acute myeloid leukemia (AML), myelogenous leukemia, chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), myeloproliferative disorder/neoplasm, myelodysplastic syndrome, monocytic cell leukemia, and plasma cell leukemia; wherein said lymphoma is selected from the group consisting of histiocytic lymphoma and T-cell lymphoma, B cell lymphomas, including Hodgkin's lymphoma and non-Hodgkin's lymphoma, such as low grade/follicular non-Hodgkin's lymphoma (NHL), cell lymphoma (FCC), mantle cell lymphoma (MCL), diffuse large cell lymphoma (DLCL), small lymphocytic (SL) NHL, intermediate grade/follicular NHL, intermediate grade diffuse NHL, high grade immunoblastic NHL, high grade lymphoblastic NHL, high grade small non-cleaved cell NHL, bulky disease NHL, and Waldenstrom's Macroglobulinemia; and wherein said sarcoma is selected from the group consisting of osteosarcoma, Ewing's sarcoma, leiomyosarcoma, synovial sarcoma, alveolar soft part sarcoma, angiosarcoma, liposarcoma, fibrosarcoma, rhabdomyosarcoma, and chrondrosarcoma.

[0110] In some embodiments, a method is disclosed to assay SIRP.alpha. expression in tumor and/or immune cells using an anti-SIRP.alpha. monoclonal antibody or antigen-binding fragment thereof, which specifically binds to an epitope within the sequence of SEQ ID NO:121.

[0111] In some embodiments, the method comprises obtaining a patient sample, contacting the patient sample with an anti-SIRP.alpha. monoclonal antibody or antigen-binding fragment thereof, which specifically binds to an epitope within the sequence of SEQ ID NO:121, and assaying for binding of the antibody to the patient sample, wherein binding of the antibody to the patient sample is diagnostic of SIRP.alpha. expression in a patient sample.

[0112] In some embodiments, a method is disclosed to assay SIRP.gamma. expression in tumor and or immune cells using an anti-SIRP.alpha. monoclonal antibody or antigen-binding fragment thereof, which specifically binds to an epitope within the sequence of SEQ ID NO:122.

[0113] In some embodiments, the method comprises obtaining a patient sample, contacting the patient sample with an anti-SIRP.gamma. monoclonal antibody or antigen-binding fragment thereof, which specifically binds to an epitope within the sequence of SEQ ID NO:122, and assaying for binding of the antibody to the patient sample, wherein binding of the antibody to the patient sample is diagnostic of SIRP.gamma. expression in a patient sample.

[0114] In some embodiments, the tumor is primary a cancer tumor or a metastatic cancer tumor.

[0115] In some embodiments, assaying for binding of the anti-SIRP.alpha. monoclonal antibody or antigen-binding fragment thereof to the patient sample utilizes immunohistochemistry labeling of a tissue sample, enzyme linked immunosorbent assay (ELISA), or flow cytometry.

[0116] In some embodiments, the method comprises tumor cells, and the assay comprises assaying for the binding of the anti-SIRP.alpha. monoclonal antibody or antigen-binding fragment thereof to tumor cells in the patient sample.

[0117] Further scope of the applicability of the present disclosure will become apparent from the detailed description provided below. However, it should be understood that the detailed description and specific examples, while indicating embodiments of the disclosure, are given by way of illustration only since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

[0118] The above and other aspects, features, and advantages of the present disclosure will be better understood from the following detailed descriptions taken in conjunction with the accompanying drawing(s), all of which are given by way of illustration only and are not limited in the present disclosure.

[0119] FIG. 1A-FIG. 1V. Binding of anti-SIRP antibodies to human SIRP.alpha.. Binding of anti-SIRP antibodies to recombinant human SIRP.alpha. was determined by solid-phase ELISA. High-binding ELISA plates were coated with recombinant human SIRP.alpha. and increasing concentrations of anti-SIRP antibodies were added for 1 hour. Wells were washed and then incubated with HRP-labeled secondary antibody for 1 hour followed by addition of peroxidase substrate and the absorbance at 450 nm was measured.

[0120] FIG. 2. Binding of Hybridoma Derived mAbs (SIRP1, SIRP2, and SIRP3) to THP1 cells Expressing SIRP.alpha.. Binding of SIRP1, SIRP2, and SIRP3 to THP-1 monocytic cell line was determined. Cells were incubated with increasing concentrations of antibody for 1 hr. Cells were washed and then incubated with Alexaflour 647-labelled secondary antibody for 1 hr. Cells were washed and antibody binding measured using flow cytometry.

[0121] FIG. 3A-FIG. 3V. Binding of anti-SIRP antibodies to human SIRP gamma. Binding of anti-SIRP antibodies to recombinant human SIRP gamma (SIRP.gamma.) was determined by solid-phase ELISA. High-binding ELISA plates were coated with recombinant human SIRP gamma and increasing concentrations of anti-SIRP antibodies were added for 1 hour. Wells were washed and then incubated with HRP-labeled secondary antibody for 1 hour followed by addition of peroxidase substrate and the absorbance at 450 nm was measured.

[0122] FIG. 4A-FIG. 4B. Binding of SIRP mAbs to Jurkat T cells Expressing SIRP.gamma.. Binding of SIRP1, SIRP2, SIRP3, SIRP4 and SIRP9 to Jurkat T-ALL cells was determined. Cells were incubated with increasing concentrations of antibody FIG. 4A; or 10 .mu.g/ml of the anti-SIRP antibodies for 1 hr; FIG. 4B. Cells were washed and then incubated with Alexaflour 647-labelled secondary antibody for 1 hr. Cells were washed and antibody binding measured using flow cytometry.

[0123] FIG. 5A-FIG. 5G. Blocking of human CD47/SIRP.alpha. binding by anti-SIRP antibodies. The ability of anti-SIRP antibodies to block the interaction between CD47 and recombinant human SIR.alpha. was determined by solid-phase ELISA. High-binding ELISA plates were coated with recombinant human SIRP.alpha. and increasing concentrations of anti-SIRP antibodies were added for 1 hour. Wells were washed and then incubated with an Fc tagged human CD47 for 1 hours. Wells were washed and then incubated with an HRP-labeled secondary antibody for 1 hour followed by addition of peroxidase substrate and the absorbance at 450 nm was measured.

[0124] FIG. 6A-FIG. 6H. Blocking of human CD47/SIRP.gamma. binding by anti-SIRP antibodies. The ability of anti-SIRP antibodies to block the interaction between CD47 and recombinant human SIRP.gamma. was determined by solid-phase ELISA. High-binding ELISA plates were coated with recombinant human SIRPyand increasing concentrations of anti-SIRP antibodies were added for 1 hour. Wells were washed and then incubated with an Fc tagged human CD47 for 1 hours. Wells were washed and then incubated with an HRP-labeled secondary antibody for 1 hour followed by addition of peroxidase substrate and the absorbance at 450 nm was measured.

[0125] FIG. 7A-FIG. 7B. Anti-SIRP antibodies enhance phagocytosis. Human macrophages were plated at a concentration of 3.times.10.sup.4 cells per well in a 96 well plate and allowed to adhere for 24 hours. 8.times.10.sup.4 CFSE (1 .mu.M) labeled human Jurkat T cells and increasing concentrations of anti-SIRP antibodies; FIG. 7A or 10 .mu.g/ml of the anti-SIRP antibodies, FIG. 7B, were added to the macrophage cultures and incubated at 37.degree. C. for 3 hours. Non-phagocytosed Jurkat cells were removed and macrophage cultures were washed. Macrophages were trypsinized and stained for CD14. Flow cytometry was used to determine the percentage of CD14.sup.+/CFSE.sup.+ cells in the total CD14.sup.+ population.

[0126] FIG. 8A-FIG. 8J. Anti-SIRP antibodies enhance phagocytosis in combination with anti-CD47 antibodies. Human macrophages were plated at a concentration of 3.times.10.sup.4 cells per well in a 96 well plate and allowed to adhere for 24 hours. 8.times.10.sup.4 CFSE (1 .mu.M) labeled human Jurkat T cells and increasing concentrations of anti-SIRP antibodies alone, anti-CD47 antibody alone, or a combination of anti-SIRP antibodies and anti-CD47 antibody were added to the macrophage cultures and incubated at 37.degree. C. for 3 hours. Non-phagocytosed Jurkat cells were removed and macrophage cultures were washed. Macrophages were trypsinized and stained for CD14. Flow cytometry was used to determine the percentage of CD14.sup.+/CFSE.sup.+ cells in the total CD14.sup.+ population.

[0127] FIG. 9A-FIG. 9D. Anti-SIRP antibodies enhance phagocytosis in combination with anti-CD20 antibodies. Human macrophages were plated at a concentration of 3.times.10.sup.4 cells per well in a 96 well plate and allowed to adhere for 24 hours. 8.times.10.sup.4 CFSE (1 .mu.M) labeled human RAJI lymphoma cells and increasing concentrations of anti-SIRP antibodies alone, the anti-CD20 antibody Rituxan alone, or a combination of anti-SIRP antibodies and Rituxan were added to the macrophage cultures and incubated at 37.degree. C. for 3 hours. Non-phagocytosed RAJI cells were removed and macrophage cultures were washed. Macrophages were trypsinized and stained for CD14. Flow cytometry was used to determine the percentage of CD14.sup.+/CFSE.sup.+ cells in the total CD14.sup.+ population.

[0128] FIG. 10A-FIG. 10B. Anti-SIRP antibodies enhance phagocytosis in combination with anti-EGFR and anti-PD-L1 antibodies. Human macrophages were plated at a concentration of 3.times.10.sup.4 cells per well in a 96 well plate and allowed to adhere for 24 hours. 8.times.10.sup.4 CFSE (1 .mu.M) labeled human FaDu HNSCC and increasing concentrations of anti-SIRP antibodies alone, the anti-EGFR antibody Erbitux alone, or anti-SIRP antibodies in combination with Erbitux or in combination with Avelumab were added to the macrophage cultures and incubated at 37.degree. C. for 3 hours. Non-phagocytosed FaDu cells were removed and macrophage cultures were washed. Macrophages were trypsinized and stained for CD14. Flow cytometry was used to determine the percentage of CD14.sup.+/CFSE.sup.+ cells in the total CD14.sup.+ population.

[0129] FIG. 11. Anti-SIRP antibodies bind to SIRP.alpha. on macrophages and dendritic cells. Binding of anti-SIRP antibodies to human macrophages or dendritic cells was determined. Human monocyte-derived macrophages were incubated with increasing concentrations of anti-SIRP antibodies for 1 hr. The cells were washed and then incubated with AF647-labelled secondary antibody for 45 min, washed and antibody binding measured using flow cytometry.

[0130] FIG. 12A-FIG. 12C. Anti-SIRP antibodies bind to SIRP.gamma. on naive and activated T cells. Binding of anti-SIRP antibodies to naive T cells (FIG. 12A and FIG. 12B) or activated T cells (FIG. 12C) following 3-day activation on anti-CD3 coated plates was determined by flow cytometry. T cells were incubated with increasing concentrations of anti-SIRP antibodies for 1 h, cells were washed and FITC-labelled anti-mouse secondary antibody was added for 1 hr. Cells were washed and antibody binding measured using flow cytometry.

[0131] FIG. 13. Blocking of human CD47/SIRP.alpha. binding by anti-SIRP antibodies on macrophages. The ability of anti-SIRP antibodies to block the interaction between recombinant human CD47 and macrophage expressed SIRP.alpha. was determined by flow cytometry. The Fc receptors on macrophages were blocked prior to incubation with 10 .mu.g/ml of the anti-SIRP antibodies. Binding of soluble Fc tagged human CD47 (20 .mu.g/ml) was measured using AF647-tagged anti-human secondary antibody.

[0132] FIG. 14A-FIG. 14B. Anti-SIRP antibodies do not inhibit T cell proliferation upon allogeneic dendritic cell stimulation. Effect of anti-SIRP antibodies on proliferation of T cells was determined by activating CellTrace Violet labelled human CD3 T cells with allogeneic human monocyte-derived dendritic cells at a 1:5 T cell:DC ratio in the presence of 10 .mu.g/ml anti-SIRP antibodies. Flow cytometry was used to determine the percentage of proliferated CD3 T cells following 6-7-day co-culture. The dotted line represents proliferation of hIgG4P control.

[0133] FIG. 15. Anti-SIRP antibodies do not inhibit antigen recall response. Effect of anti-SIRP antibodies on T cell antigen recall responses was assessed using PBMC from human cytomegalovirus seropositive donor. CellTrace Violet dye-labelled PBMC were incubated with 10 .mu.g/ml of anti-SIRP antibodies in the presence of increasing concentrations of CMV antigen for 5 days. T cell proliferation was determined by the dilution of the CellTrace Violet dye within the CD4+ T cell population using flow cytometry.

DETAILED DESCRIPTION OF THE DISCLOSURE

Definitions

[0134] Unless otherwise defined, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Generally, nomenclatures utilized in connection with, and techniques of, cell and tissue culture, molecular biology, and protein and oligo or polynucleotide chemistry and hybridization described herein are those well-known and commonly used in the art.

[0135] As used herein, the term "SIRP.alpha." and "Src homology 2 (SH2) domain-containing protein tyrosine phosphatase substrate 1 (SHPS-1)" are synonymous and may be used interchangeably.

[0136] The term "anti-SIRP.alpha. antibody" refer to an antibody of the disclosure which is intended for use as a therapeutic or diagnostic agent, and specifically binds to SIRP.alpha., in particular to a human SIRP.alpha..

[0137] The term "anti-SIRP" refer to an antibody of the disclosure which is intended for use as a therapeutic or diagnostic agent, and specifically binds to SIRP.alpha., in particular to a human SIRP.alpha., to one or both of two common variants identified, SIRP.alpha.V1 and SIRP.alpha.V2, and/or SIRP.gamma. and antibody variants thereof.

[0138] As used herein, the term "antibody" refers to immunoglobulin molecules and immunologically active portions of immunoglobulin (Ig) molecules, i.e., molecules that contain an antigen binding site that specifically binds (immunoreacts with) an antigen. By "specifically bind" or "immunoreacts" with or directed against is meant that the antibody reacts with one or more antigenic determinants of the desired antigen and does not react with other polypeptides or binds at a much lower affinity (K.sub.d>10.sup.-6 M). Antibodies include but are not limited to, polyclonal, monoclonal, chimeric, Fab fragments, Fab' fragments, F(ab').sub.2 fragments, single chain Fv fragments, and one-armed antibodies.

[0139] As used herein, the term "monoclonal antibody (mAb)" as applied to the present anti-SIRP.alpha. compounds refer to an antibody that is derived from a single copy or clone including, for example, any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced. Monoclonal antibodies of the present disclosure preferably exist in a homogeneous or substantially homogeneous population. Complete mAbs contain 2 heavy-chains and 2 light-chains.

[0140] An "antibody fragment" refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include but are not limited to Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g. scFv); and multi-specific antibodies formed from antibody fragments.

[0141] As disclosed herein, "antibody compounds" refers to mAbs and antigen-binding fragments thereof. Additional antibody compounds exhibiting similar functional properties according to the present disclosure can be generated by conventional methods. For example, mice can be immunized with human SIRP.alpha. or fragments thereof, the resulting antibodies can be recovered and purified, and determination of whether they possess binding and functional properties similar to or the same as the antibody compounds disclosed herein can be assessed by the methods disclosed in the Examples. Antigen-binding fragments can also be prepared by conventional methods. Methods for producing and purifying antibodies and antigen-binding fragments are well known in the art and can be found, for example, in Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., chapters 5-8 and 15.

[0142] As disclosed herein, "multispecific antibodies" are e.g., bispecific, trispecific or tetraspecific antibodies. In some embodiments, the multispecific antibodies target SIRP.alpha. and/or SIRP.gamma. and at least one other antigen binding specificity in one molecule. In some embodiments, the multispecific antibodies may simultaneously target SIRP.alpha. and/or SIRP.gamma. and at least a second antigen (bispecific), or at least a second and third antigen (trispecific), or at least a second, third, and fourth antigen (tetraspecific), wherein the second antigen, third antigen, and fourth antigen is on a tumor cell as disclosed herein.

[0143] Bispecific antibodies are antibodies which have two different antigen binding specificities in one molecule. Trispecific antibodies, accordingly, are antibodies which have three different antigen-binding specificities in one molecule. Tetraspecific antibodies are antibodies which have four different antigen-binding specificities in one molecule. In one embodiment, the anti-SIRP.alpha. antibodies as disclosed herein are bispecific antibodies targeting SIRP.alpha. and/or SIRP.gamma., and a second antigen on a tumor cell as disclosed herein.

[0144] The monoclonal antibodies encompass antibodies in which a portion of the heavy and/or light-chain is identical with, or homologous to, corresponding sequences in murine antibodies, in particular the murine CDRs, while the remainder of the chain(s) is (are) identical with, or homologous to, corresponding sequences in human antibodies. Other embodiments of the disclosure include antigen-binding fragments of these monoclonal antibodies that exhibit binding and biological properties similar or identical to the monoclonal antibodies. The antibodies of the present disclosure can comprise kappa or lambda light-chain constant regions, and heavy-chain IgA, IgD, IgE, IgG, or IgM constant regions, including those of IgG subclasses IgG1, IgG2, IgG3, and IgG4 and in some cases with various mutations to alter Fc receptor function.

[0145] The monoclonal antibodies containing the presently disclosed murine CDRs can be prepared by any of the various methods known to those skilled in the art, including recombinant DNA methods.

[0146] Reviews of current methods for antibody engineering and improvement can be found, for example, in P. Chames, Ed., (2012) Antibody Engineering: Methods and Protocols, Second Edition (Methods in Molecular Biology, Book 907), Humana Press, ISBN-10: 1617799734; C. R. Wood, Ed., (2011) Antibody Drug Discovery (Molecular Medicine and Medicinal Chemistry, Book 4), Imperial College Press; R. Kontermann and S. Dubel, Eds., (2010) Antibody Engineering Volumes 1 and 2 (Springer Protocols), Second Edition; and W. Strohl and L. Strohl (2012) Therapeutic antibody engineering: Current and future advances driving the strongest growth area in the pharmaceutical industry, Woodhead Publishing.

[0147] Methods for producing and purifying antibodies and antigen-binding fragments are well known in the art and can be found, for example, in Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., chapters 5-8 and 15.

[0148] A full-length antibody as it exists naturally is a "Y" shaped immunoglobulin (Ig) molecule comprising four polypeptide chains: two identical heavy (H) chains and two identical light (L) chains, interconnected by disulfide bonds. The amino terminal portion of each chain, termed the fragment antigen binding region (FAB), includes a variable region of about 100-110 or more amino acids primarily responsible for antigen recognition via the complementarity determining regions (CDRs) contained therein. The carboxy-terminal portion of each chain defines a constant region (the "Fc" region) primarily responsible for effector function.

[0149] The CDRs are interspersed with regions that are more conserved, termed frameworks ("FRs"). Amino acid sequences of many FRs are well known in the art. Each light-chain variable region (LCVR) and heavy-chain variable region (HCVR) is composed of 3 CDRs and 4 FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The 3 CDRs of the light-chain are referred to as "LCDR1, LCDR2, and LCDR3" and the 3 CDRs of the heavy-chain are referred to as "HCDR1, HCDR2, and HCDR3." The CDRs contain most of the residues which form specific interactions with the antigen. The numbering and positioning of CDR amino acid residues within the LCVR and HCVR regions are in accordance with the well-known Kabat numbering convention Kabat et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition. NIH Publication No. 91-3242.

[0150] As described herein, the "antigen-binding site" can also be defined as the "Hypervariable regions", "HVRs", or "HVs", and refer to the structurally hypervariable regions of antibody variable domains as defined by Chothia and Lesk (Chothia and Lesk, Mol. Biol. 196:901-917, 1987). There are six HVRs, three in VH (H1, H2, H3) and three in VL (L1, L2, L3). CDRs as defined by Kabat were used herein except in H-CDR1, which is extended to include H1.

[0151] There are five types of mammalian immunoglobulin (Ig) heavy-chains, denoted by the Greek letters .alpha. (alpha), .delta. (delta), .epsilon. (epsilon), .gamma. (gamma), and .mu. (mu), which define the class or isotype of an antibody as IgA, IgD, IgE, IgG, or IgM, respectively. IgG antibodies can be further divided into subclasses, for example, IgG1, IgG2, IgG3, and IgG4.

[0152] Each heavy-chain type is characterized by a particular constant region with a sequence well known in the art. The constant region is identical in all antibodies of the same isotype but differs in antibodies of different isotypes. Heavy-chains .gamma., .alpha., and .delta. have a constant region composed of three tandem immunoglobulin (Ig) domains, and a hinge region for added flexibility. Heavy-chains .mu. and .epsilon. have a constant region composed of four Ig domains.

[0153] The hinge region is a flexible amino acid stretch that links the Fc and Fab portions of an antibody. This region contains cysteine residues that can form disulfide bonds, connecting two heavy-chains together.

[0154] The variable region of the heavy-chain differs in antibodies produced by different B cells but is the same for all antibodies produced by a single B cell or B cell clone. The variable region of each heavy-chain is approximately 110 amino acids long and is composed of a single Ig domain.

[0155] In mammals, light-chains are classified as kappa (.kappa.) or lambda (.lamda.), and are characterized by a particular constant region as known in the art. A light-chain has two successive domains: one variable domain at the amino-terminal end, and one constant domain at the carboxy-terminal end. Each antibody contains two light-chains that are always identical; only one type of light-chain, .kappa. or .lamda., is present per antibody in mammals.

[0156] The Fc region, composed of two heavy-chains that contribute three or four constant domains depending on the class of the antibody, plays a role in modulating immune cell activity. By binding to specific proteins, the Fc region ensures that each antibody generates an appropriate immune response for a given antigen. The Fc region also binds to various cell receptors, such as Fc receptors, and other immune molecules, such as complement proteins. By doing this, it mediates different physiological effects, including opsonization, cell lysis, and degranulation of mast cells, basophils and eosinophils.

[0157] As used herein, the term "epitope" refers to a specific arrangement of amino acids located on a peptide or protein to which an antibody or antibody fragment binds. Epitopes often consist of a chemically active surface grouping of molecules such as amino acids or sugar side chains and have specific three-dimensional structural characteristics as well as specific charge characteristics. Epitopes can be linear, i.e., involving binding to a single sequence of amino acids, or conformational, i.e., involving binding to two or more sequences of amino acids in various regions of the antigen that may not necessarily be contiguous in the linear sequence.

[0158] As used herein, the terms "specifically binds", "bind specifically", "specific binding", and the like as applied to the present antibody compounds refer to the ability of a specific binding agent (such as an antibody) to bind to a target molecular species in preference to binding to other molecular species with which the specific binding agent and target molecular species are admixed. A specific binding agent is said specifically to recognize a target molecular species when it can bind specifically to that target.

[0159] As used herein, the term "binding affinity" refers to the strength of binding of one molecule to another at a site on the molecule. If a particular molecule will bind to or specifically associate with another particular molecule, these two molecules are said to exhibit binding affinity for each other. Binding affinity is related to the association constant and dissociation constant for a pair of molecules, but it is not critical to the methods herein that these constants be measured or determined. Rather, affinities as used herein to describe interactions between molecules of the described methods are generally apparent affinities (unless otherwise specified) observed in empirical studies, which can be used to compare the relative strength with which one molecule (e.g., an antibody or other specific binding partner) will bind two other molecules (e.g., two versions or variants of a peptide). The concepts of binding affinity, association constant, and dissociation constant are well known.

[0160] As used herein, the term "sequence identity" means the percentage of identical nucleotide or amino acid residues at corresponding positions in two or more sequences when the sequences are aligned to maximize sequence matching, i.e., considering gaps and insertions. Identity can be readily calculated by known methods, including but not limited to those described in: Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073 (1988). Methods to determine identity are designed to give the largest match between the sequences tested. Moreover, methods to determine identity are codified in publicly available computer programs.

[0161] Optimal alignment of sequences for comparison can be conducted, for example, by the local homology algorithm of Smith & Waterman, by the homology alignment algorithms, by the search for similarity method or, by computerized implementations of these algorithms (GAP, BESTFIT, PASTA, and TFASTA in the GCG Wisconsin Package, available from Accelrys, Inc., San Diego, Calif., United States of America), or by visual inspection. See generally, Altschul, S. F. et al., J. Mol. Biol. 215: 403-410 (1990) and Altschul et al. Nucl. Acids Res. 25: 3389-3402 (1997).

[0162] One example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in (Altschul, S., et al., NCBI NLM NIH Bethesda, Md. 20894; and Altschul, S., et al., J. Mol. Biol. 215: 403-410 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold.

[0163] These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always; 0) and N (penalty score for mismatching residues; always; 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value, the cumulative score goes to zero or below due to the accumulation of one or more negative-scoring residue alignments, or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 11, an expectation (E) of 10, a cutoff of 100, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word length (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix.

[0164] In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences. One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a test nucleic acid sequence is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid sequence to the reference nucleic acid sequence is in one embodiment less than about 0.1, in another embodiment less than about 0.01, and in still another embodiment less than about 0.001.

[0165] As used herein, the terms "humanized", "humanization", and the like, refer to grafting of the murine monoclonal antibody CDRs disclosed herein to human FRs and constant regions. Also encompassed by these terms are possible further modifications to the murine CDRs, and human FRs, by the methods disclosed in, for example, Kashmiri et al. (2005) Methods 36(1):25-34 and Hou et al. (2008) J. Biochem. 144(1):115-120, respectively, to improve various antibody properties, as discussed below.

[0166] As used herein, the term "humanized antibodies" refers to mAbs and antigen binding fragments thereof, including the antibody compounds disclosed herein, that have binding and functional properties according to the disclosure similar to those disclosed herein, and that have FRs and constant regions that are substantially human or fully human surrounding CDRs derived from a non-human antibody.

[0167] As used herein, the term "FR" or "framework sequence" refers to any one of FRs 1 to 4. Humanized antibodies and antigen binding fragments encompassed by the present disclosure include molecules wherein any one or more of FRs 1 to 4 is substantially or fully human, i.e., wherein any of the possible combinations of individual substantially or fully human FRs 1 to 4, is present. For example, this includes molecules in which FR1 and FR2, FR1 and FR3, FR1, FR2, and FR3, etc., are substantially or fully human. Substantially human frameworks are those that have at least 80% sequence identity to a known human germline framework sequence. Preferably, the substantially human frameworks have at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity, to a framework sequence disclosed herein, or to a known human germline framework sequence.

[0168] Fully human frameworks are those that are identical to a known human germline framework sequence. Human FR germline sequences can be obtained from the international ImMunoGeneTics (IMGT) database and from The Immunoglobulin FactsBook by Marie-Paule Lefranc and Gerard Lefranc, Academic Press, 2001, the contents of which are herein incorporated by reference in their entirety.

[0169] The Immunoglobulin Facts Book is a compendium of the human germline immunoglobulin genes that are used to create the human antibody repertoire, and includes entries for 203 genes and 459 alleles, with a total of 837 displayed sequences. The individual entries comprise all the human immunoglobulin constant genes, and germline variable, diversity, and joining genes that have at least one functional or open reading frame allele, and which are localized in the three major loci. For example, germline light-chain FRs can be selected from the group consisting of: IGKV3D-20, IGKV2-30, IGKV2-29, IGKV2-28, IGKV1-27, IGKV3-20, IGKV1-17, IGKV1-16, 1-6, IGKV1-5, IGKV1-12, IGKV1D-16, IGKV2D-28, IGKV2D-29, IGKV3-11, IGKV1-9, IGKV1-39, IGKV1D-39 and IGKV1D-33 and IGKJ1-5 and germline heavy-chain FRs can be selected from the group consisting of: IGHV1-2, IGHV1-18, IGHV1-46, IGHV1-69, IGHV2-5, IGHV2-26, IGHV2-70, IGHV1-3, IGHV1-8, IGHV3-9, IGHV3-11, IGHV3-15, IGHV3-20, IGHV3-66, IGHV3-72, IGHV3-74, IGHV4-31, IGHV3-21, IGHV3-23, IGHV3-30, IGHV3-48, IGHV4-39, IGHV4-59 and IGHV5-51 and IGHJ1-6.

[0170] Substantially human FRs are those that have at least 80% sequence identity to a known human germline FR sequence. Preferably, the substantially human frameworks have at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity, to a framework sequences disclosed herein, or to a known human germline framework sequence.

[0171] CDRs encompassed by the present disclosure include not only those specifically disclosed herein, but also CDR sequences having sequence identities of at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a CDR sequence disclosed herein. Alternatively, CDRs encompassed by the present disclosure include not only those specifically disclosed herein, but also CDR sequences having 1, 2, 3, 4, or 5 amino acid changes at corresponding positions compared to CDR sequences disclosed herein. Such sequence identical, or amino acid modified, CDRs preferably bind to the antigen recognized by the intact antibody.

[0172] Humanized antibodies in addition to those disclosed herein exhibiting similar functional properties according to the present disclosure can be generated using several different methods Almagro et al. Frontiers in Biosciences. Humanization of antibodies. (2008) Jan. 1; 13:1619-33. In one approach, the parent antibody compound CDRs are grafted into a human framework that has a high sequence identity with the parent antibody compound framework. The sequence identity of the new framework will generally be at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identical to the sequence of the corresponding framework in the parent antibody compound. In the case of frameworks having fewer than 100 amino acid residues, one, two, three, four, five, six, seven, eight, nine, or ten amino acid residues can be changed. This grafting may result in a reduction in binding affinity compared to that of the parent antibody. If this is the case, the framework can be back-mutated to the parent framework at certain positions based on specific criteria disclosed by Queen et al. (1991) Proc. Natl. Acad. Sci. USA 88:2869. Additional references describing methods useful to generate humanized variants based on homology and back mutations include as described in Olimpieri et al. Bioinformatics. 2015 Feb. 1; 31(3):434-435 and U.S. Pat. Nos. 4,816,397, 5,225,539, and 5,693,761; and the method of Winter and co-workers (Jones et al. (1986) Nature 321:522-525; Riechmann et al. (1988) Nature 332:323-327; and Verhoeyen et al. (1988) Science 239:1534-1536.

[0173] Humanization began with chimerization, a method developed during the first half of the 1980's (Morrison, S. L., M. J. Johnson, L. A. Herzenberg & V. T. Oi: Chimeric human antibody molecules: mouse antigen-binding domains with human constant region domains. Proc. Natl. Acad. Sci. USA., 81, 6851-5 (1984)), consisting of combining the variable (V) domains of murine antibodies with human constant (C) domains to generate molecules with .about.70% of human content.

[0174] Several different methods can be used to generate humanized antibodies, which are described herein. In one approach, the parent antibody compound CDRs are grafted into a human FR that has a high sequence identity with the parent antibody compound framework. The sequence identity of the new FR will generally be at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence of the corresponding FR in the parent antibody compound. In the case of FRs having fewer than 100 amino acid residues, one, two, three, four, five, or more amino acid residues can be changed. This grafting may result in a reduction in binding affinity compared to that of the parent antibody. If this is the case, the FR can be back-mutated to the parent framework at certain positions based on specific criteria disclosed by Queen et al. (1991) Proc. Natl. Acad. Sci. USA 88:2869. Additional references describing methods useful to generate humanized variants based on homology and back mutations include as described in Olimpieri et al. Bioinformatics. 2015 Feb. 1; 31(3):434-435 and U.S. Pat. Nos. 4,816,397, 5,225,539, and 5,693,761; and the method of Winter and co-workers (Jones et al. (1986) Nature 321:522-525; Riechmann et al. (1988) Nature 332:323-327; and Verhoeyen et al. (1988) Science 239:1534-1536.

[0175] The identification of residues to consider for back-mutation can be carried out as described below. When an amino acid falls under the following category, the framework amino acid of the human germ-line sequence that is being used (the "acceptor FR") is replaced by a framework amino acid from a framework of the parent antibody compound (the "donor FR"): [0176] (a) the amino acid in the human FR of the acceptor framework is unusual for human frameworks at that position, whereas the corresponding amino acid in the donor immunoglobulin is typical for human frameworks at that position; [0177] (b) the position of the amino acid is immediately adjacent to one of the CDRs; or [0178] (c) any side chain atom of a framework amino acid is within about 5-6 angstroms (center-to-center) of any atom of a CDR amino acid in a three-dimensional immunoglobulin model.

[0179] When each of the amino acids in the human FR of the acceptor framework and a corresponding amino acid in the donor framework is generally unusual for human frameworks at that position, such amino acid can be replaced by an amino acid typical for human frameworks at that position. This back-mutation criterion enables one to recover the activity of the parent antibody compound.

[0180] Another approach to generating humanized antibodies exhibiting similar functional properties to the antibody compounds disclosed herein involves randomly mutating amino acids within the grafted CDRs without changing the framework and screening the resultant molecules for binding affinity and other functional properties that are as good as, or better than, those of the parent antibody compounds. Single mutations can also be introduced at each amino acid position within each CDR, followed by assessing the effects of such mutations on binding affinity and other functional properties. Single mutations producing improved properties can be combined to assess their effects in combination with one another.

[0181] Further, a combination of both of the foregoing approaches is possible. After CDR grafting, one can back-mutate specific FRs in addition to introducing amino acid changes in the CDRs. This methodology is described in Wu et al. (1999) J. Mol. Biol. 294: 151-162.

[0182] Applying the teachings of the present disclosure, a person skilled in the art can use common techniques, e.g., site-directed mutagenesis, to substitute amino acids within the presently disclosed CDR and FR sequences and thereby generate further variable region amino acid sequences derived from the present sequences. Up to all naturally occurring amino acids can be introduced at a specific substitution site. The methods disclosed herein can then be used to screen these additional variable region amino acid sequences to identify sequences having the indicated in vivo functions. In this way, further sequences suitable for preparing humanized antibodies and antigen-binding portions thereof in accordance with the present disclosure can be identified. Preferably, amino acid substitution within the frameworks is restricted to one, two, three, four, or five positions within any one or more of the four light-chain and/or heavy-chain FRs disclosed herein. Preferably, amino acid substitution within the CDRs is restricted to one, two, three, four, or five positions within any one or more of the three light-chain and/or heavy-chain CDRs. Combinations of the various changes within these FRs and CDRs described above are also possible.

[0183] That the functional properties of the antibody compounds generated by introducing the amino acid modifications discussed above conform to those exhibited by the specific molecules disclosed herein can be confirmed by the methods in Examples disclosed herein.

[0184] As described above, to circumvent the problem of eliciting human anti-murine antibody (HAMA) response in patients, murine antibodies have been genetically manipulated to progressively replace their murine content with the amino acid residues present in their human counterparts by grafting their complementarity determining regions (CDRs) onto the variable light (V.sub.L) and variable heavy (V.sub.H) frameworks of human immunoglobulin molecules, while retaining those murine framework residues deemed essential for the integrity of the antigen-combining site. However, the xenogeneic CDRs of the humanized antibodies may evoke anti-idiotypic (anti-Id) response in patients.

[0185] To minimize the anti-Id response, a procedure to humanize xenogeneic antibodies by grafting onto the human frameworks only the CDR residues most crucial in the antibody-ligand interaction, called "SDR grafting", has been developed, wherein only the crucial specificity determining residues (SDRs) of CDRS are grafted onto the human frameworks. This procedure, described in Kashmiri et al. (2005) Methods 36(1):25-34, involves identification of SDRs through the help of a database of the three-dimensional structures of the antigen-antibody complexes of known structures, or by mutational analysis of the antibody-combining site. An alternative approach to humanization involving retention of more CDR residues is based on grafting of the `abbreviated` CDRs, the stretches of CDR residues that include all the SDRs. Kashmiri et al. also discloses a procedure to assess the reactivity of humanized antibodies to sera from patients who had been administered the murine antibody.

[0186] Another strategy for constructing human antibody variants with improved immunogenic properties is disclosed in Hou et al. (2008) J. Biochem. 144(1):115-120. These authors developed a humanized antibody from 4C8, a murine anti-human CD34 monoclonal antibody, by CDR grafting using a molecular model of 4C8 built by computer-assisted homology modelling. Using this molecular model, the authors identified FR residues of potential importance in antigen binding. A humanized version of 4C8 was generated by transferring these key murine FR residues onto a human antibody framework that was selected based on homology to the murine antibody FR, together with the murine CDR residues. The resulting humanized antibody was shown to possess antigen-binding affinity and specificity similar to that of the original murine antibody, suggesting that it might be an alternative to murine anti-CD34 antibodies routinely used clinically.

[0187] Embodiments of the present disclosure encompass antibodies created to avoid recognition by the human immune system containing CDRs disclosed herein in any combinatorial form such that contemplated mAbs can contain the set of CDRs from a single murine mAb disclosed herein, or light and heavy-chains containing sets of CDRs comprising individual CDRs derived from two or three of the disclosed murine mAbs. Such mAbs can be created by standard techniques of molecular biology and screened for desired activities using assays described herein. In this way, the disclosure provides a "mix and match" approach to create novel mAbs comprising a mixture of CDRs from the disclosed murine mAbs to achieve new, or improved, therapeutic activities.

[0188] Monoclonal antibodies or antigen-binding fragments thereof encompassed by the present disclosure that "compete" with the molecules disclosed herein are those that bind human SIRP.alpha. at site(s) that are identical to, or overlapping with, the site(s) at which the present molecules bind. Competing monoclonal antibodies or antigen-binding fragments thereof can be identified, for example, via an antibody competition assay. For example, a sample of purified or partially purified human SIRP.alpha. extracellular domain can be bound to a solid support. Then, an antibody compound, or antigen binding fragment thereof, of the present disclosure and a monoclonal antibody or antigen-binding fragment thereof suspected of being able to compete with such disclosure antibody compound are added. One of the two molecules is labeled. If the labeled compound and the unlabeled compound bind to separate and discrete sites on SIRP.alpha., the labeled compound will bind to the same level whether or not the suspected competing compound is present. However, if the sites of interaction are identical or overlapping, the unlabeled compound will compete, and the amount of labeled compound bound to the antigen will be lowered. If the unlabeled compound is present in excess, very little, if any, labeled compound will bind. For purposes of the present disclosure, competing monoclonal antibodies or antigen-binding fragments thereof are those that decrease the binding of the present antibody compounds to SIRP.alpha. by about 50%, about 60%, about 70%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%. Details of procedures for carrying out such competition assays are well known in the art and can be found, for example, in Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. Such assays can be made quantitative by using purified antibodies. A standard curve is established by titrating one antibody against itself, i.e., the same antibody is used for both the label and the competitor. The capacity of an unlabeled competing monoclonal antibody or antigen-binding fragment thereof to inhibit the binding of the labeled molecule to the plate is titrated. The results are plotted, and the concentrations necessary to achieve the desired degree of binding inhibition are compared.

[0189] Whether mAbs or antigen-binding fragments thereof that compete with antibody compounds of the present disclosure in such competition assays possess the same or similar functional properties of the present antibody compounds can be determined via these methods in conjunction with the methods described in Examples 2-7, below. In various embodiments, competing antibodies for use in the therapeutic methods encompassed herein possess biological activities as described herein in the range of from about 50% to about 100% or about 125%, or more, compared to that of the antibody compounds disclosed herein. In some embodiments, competing antibodies possess about 50%, about 60%, about 70%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or identical biological activity compared to that of the antibody compounds disclosed herein as determined by the methods disclosed in the Examples presented below.

[0190] The mAbs or antigen-binding fragments thereof or competing antibodies useful in the compositions and methods can be any of the isotypes described herein. Furthermore, any of these isotypes can comprise further amino acid modifications as follows.

[0191] The monoclonal antibody or antigen-binding fragment thereof, or competing antibody described herein can be of the human IgG1 isotype.

[0192] The human IgG1 constant region of the monoclonal antibody, antigen-binding fragment thereof, or competing antibody described herein can be modified to alter antibody half-life. Antibody half-life is regulated in large part by Fc-dependent interactions with the neonatal Fc receptor (Roopenian and Alikesh, 2007). The human IgG1 constant region of the monoclonal antibody, antigen-binding fragment thereof, or competing antibody can be modified to increase half-life include, but are not limited to amino acid modifications N434A, T307A/E380A/N434A (Petkova et al., 2006, Yeung et al., 2009); M252Y/S254T/T256E (Dall'Acqua et al., 2006); T250Q/M428L (Hinton et al., 2006); and M428L/N434S (Zalevsky et al., 2010).

[0193] As opposed to increasing half-life, there are some circumstances where decreased half-life would be desired, such as to reduce the possibility of adverse events associated with high Antibody-Dependent Cellular Cytotoxicity (ADCC) and Complement-Dependent Cytotoxicity (CDC) antibodies (Presta 2008). The human IgG1 constant region of the monoclonal antibody, antigen-binding fragment thereof, or competing antibody described herein can be modified to decrease half-life and/or decrease endogenous IgG include, but are not limited to, amino acid modifications I253A (Petkova et al., 2006); P257I/N434H, D376V/N434H (Datta-Mannan et al., 2007); and M252Y/S254T/T256E/H433K/N434F (Vaccaro et al., 2005).

[0194] The human IgG1 constant region of the monoclonal antibody, antigen-binding fragment thereof, or competing antibody described herein can be modified to increase or decrease antibody effector functions. These antibody effector functions include, but are not limited to, Antibody-Dependent Cellular Cytotoxicity (ADCC), Complement-Dependent Cytotoxicity (CDC), Antibody-Dependent Cellular Phagocytosis (ADCP), C1q binding, and altered binding to Fc receptors.

[0195] The human IgG1 constant region of the monoclonal antibody, antigen-binding fragment thereof, or competing antibody described herein can be modified to increase antibody effector function include, but are not limited to amino acid modifications S298A/E333A/K334 (Shields et al., 2001); S239D/I332E and S239D/A330L/I332E (Lazar et al., 2006); F234L/R292P/Y300L, F234L/R292P/Y300L/P393L, and F243L/R292P/Y300L/V305I/P396L (Stevenhagen et al., 2007); G236A, G236A/S239D/I332E, and G236A/S239D/A330L/I332E (Richards et al., 2008); K326A/E333A, K326A/E333S and K326W/E333S (Idusogie et al., 2001); S267E and S267E/L328F (Smith et al., 2012); H268F/S324T, S267E/H268F, S267E/S234T, and S267E/H268F/S324T (Moore et al., 2010); S298G/T299A (Sazinsky et al., 2008); E382V/M428I (Jung et al., 2010).

[0196] The human IgG1 constant region of the monoclonal antibody, antigen-binding fragment thereof, or competing antibody described herein can be modified to decrease antibody effector function include, but are not limited to amino acid modifications N297A and N297Q (Bolt et al., 1993, Walker et al., 1989); L234A/L235A (Xu et al., 2000); K214T/E233P/L234V/L235A/G236-deleted/A327G/P331A/D356E/L358M (Ghevaert et al., 2008); C226S/C229S/E233P/L234V/L235A (McEarchern et al., 2007); S267E/L328F (Chu et al., 2008).

[0197] The human IgG1 constant region of the monoclonal antibody, antigen-binding fragment thereof, or competing antibody described herein can be modified to decrease antibody effector function include, but are not limited to amino acid modifications V234A/G237A (Cole et al., 1999); E233D, G237D, P238D, H268Q, H268D, P271G, V309L, A330S, A330R, P331S, H268Q/A330S/V309L/P331S, H268D/A330S/V309L/P331S, H268Q/A330R/V309L/P331S, H268D/A330R/V309L/P331S, E233D/A330R, E233D/A330S, E233D/P271G/A330R, E233D/P271G/A330S, G237D/H268D/P271G, G237D/H268Q/P271G, G237D/P271G/A330R, G237D/P271G/A330S, E233D/H268D/P271G/A330R, E233D/H268Q/P271G/A330R, E233D/H268D/P271G/A330S, E233D/H268Q/P271G/A330S, G237D/H268D/P271G/A330R, G237D/H268Q/P271G/A330R, G237D/H268D/P271G/A330S, G237D/H268Q/P271G/A330S, E233D/G237D/H268D/P271G/A330R, E233D/G237D/H268Q/P271G/A330R, E233D/G237D/H268D/P271G/A330S, E233D/G237D/H268Q/P271G/A330S, P238D/E233D/A330R, P238D/E233D/A330S, P238D/E233D/P271G/A330R, P238D/E233D/P271G/A330S, P238D/G237D/H268D/P271G, P238D/G237D/H268Q/P271G, P238D/G237D/P271G/A330R, P238D/G237D/P271G/A330S, P238D/E233D/H268D/P271G/A330R, P238D/E233D/H268Q/P271G/A330R, P238D/E233D/H268D/P271G/A330S, P238D/E233D/H268Q/P271G/A330S, P238D/G237D/H268D/P271G/A330R, P238D/G237D/H268Q/P271G/A330R, P238D/G237D/H268D/P271G/A330S, P238D/G237D/H268Q/P271G/A330S, P238D/E233D/G237D/H268D/P271G/A330R, P238D/E233D/G237D/H268Q/P271G/A330R, P238D/E233D/G237D/H268D/P271G/A330S, P238D/E233D/G237D/H268Q/P271G/A330S (An et al., 2009, Mimoto, 2013).

[0198] The monoclonal antibody or antigen-binding fragment thereof, or competing antibody described herein can be of the human IgG2 isotype.

[0199] The human IgG2 constant region of the monoclonal antibody, antigen-binding fragment thereof, or competing antibody described herein can be modified to increase or decrease antibody effector functions. These antibody effector functions include, but are not limited to, Antibody-Dependent Cellular Cytotoxicity (ADCC), Complement-Dependent Cytotoxicity (CDC), Antibody-Dependent Cellular Phagocytosis (ADCP), and C1q binding, and altered binding to Fc receptors.

[0200] The human IgG2 constant region of the monoclonal antibody, antigen-binding fragment thereof, or a competing antibody described herein, can be modified to increase antibody effector function include, but are not limited to, the amino acid modification K326A/E333S (Idusogie et al., 2001).

[0201] The human IgG2 constant region of the monoclonal antibody, antigen-binding fragment thereof, or competing antibody described herein can be modified to decrease antibody effector function include, but are not limited to amino acid modifications V234A/G237A (Cole et al., 1999); E233D, G237D, P238D, H268Q, H268D, P271G, V309L, A330S, A330R, P331S, H268Q/A330S/V309L/P331S, H268D/A330S/V309L/P331S, H268Q/A330R/V309L/P331S, H268D/A330R/V309L/P331S, E233D/A330R, E233D/A330S, E233D/P271G/A330R, E233D/P271G/A330S, G237D/H268D/P271G, G237D/H268Q/P271G, G237D/P271G/A330R, G237D/P271G/A330S, E233D/H268D/P271G/A330R, E233D/H268Q/P271G/A330R, E233D/H268D/P271G/A330S, E233D/H268Q/P271G/A330S, G237D/H268D/P271G/A330R, G237D/H268Q/P271G/A330R, G237D/H268D/P271G/A330S, G237D/H268Q/P271G/A330S, E233D/G237D/H268D/P271G/A330R, E233D/G237D/H268Q/P271G/A330R, E233D/G237D/H268D/P271G/A330S, E233D/G237D/H268Q/P271G/A330S, P238D/E233D/A330R, P238D/E233D/A330S, P238D/E233D/P271G/A330R, P238D/E233D/P271G/A330S, P238D/G237D/H268D/P271G, P238D/G237D/H268Q/P271G, P238D/G237D/P271G/A330R, P238D/G237D/P271G/A330S, P238D/E233D/H268D/P271G/A330R, P238D/E233D/H268Q/P271G/A330R, P238D/E233D/H268D/P271G/A330S, P238D/E233D/H268Q/P271G/A330S, P238D/G237D/H268D/P271G/A330R, P238D/G237D/H268Q/P271G/A330R, P238D/G237D/H268D/P271G/A330S, P238D/G237D/H268Q/P271G/A330S, P238D/E233D/G237D/H268D/P271G/A330R, P238D/E233D/G237D/H268Q/P271G/A330R, P238D/E233D/G237D/H268D/P271G/A330S, P238D/E233D/G237D/H268Q/P271G/A330S (An et al., 2009, Mimoto, 2013).

[0202] The Fc region of a human IgG2 of the monoclonal antibody, antigen-binding fragment thereof, or competing antibody described herein can be modified to alter isoform and/or agonistic activity, include, but are not limited to amino acid modifications C127S (C.sub.H1 domain), C232S, C233S, C232S/C233S, C236S, and C239S (White et al., 2015, Lightle et al., 2010).

[0203] The Fc region of a human IgG2 of the monoclonal antibody, antigen-binding fragment thereof, or competing antibody described herein can be modified to exhibit diminished Fc.gamma.R binding capacity but have conserved FcRn binding. These IgG Fc mutants enable therapeutic targeting of soluble or cell surface antigens while minimizing Fc-associated engagement of immune effector function and complement mediated cytotoxicity. In one embodiment, the IgG2 Fc mutant comprises V234A, G237A, P238S according to the EU numbering system. In another embodiment, the IgG2 Fc mutant comprises V234A, G237A, H268Q, or H268A, V309L, A330S, P331S, according to the EU numbering system. In a particular aspect, the IgG2 Fc mutant comprises V234A, G237A, P238S, H268A, V309L, A330S, P331S, and, optionally, P233S according to the EU numbering system.

[0204] The monoclonal antibody or antigen-binding fragment thereof, or competing antibody described herein can be of the human IgG3 isotype.

[0205] The human IgG3 constant region of the monoclonal antibody, or antigen binding fragment thereof, wherein said human IgG3 constant region of the monoclonal antibody, or antigen-binding fragment thereof can be modified at one or more amino acid(s) to increase antibody half-life, Antibody-Dependent Cellular Cytotoxicity (ADCC), Complement-Dependent Cytotoxicity (CDC), or apoptosis activity.

[0206] The human IgG3 constant region of the monoclonal antibody, or antigen-binding fragment thereof, wherein said human IgG3 constant region of the monoclonal antibody, or antigen-binding fragment thereof can be modified at amino acid R435H to increase antibody half-life.

[0207] The monoclonal antibody or antigen-binding fragment thereof, or competing antibody described herein can be of the human IgG4 isotype.

[0208] The human IgG4 constant region of the monoclonal antibody, antigen-binding fragment thereof, or competing antibody described herein can be modified to decrease antibody effector functions. These antibody effector functions include, but are not limited to, Antibody-Dependent Cellular Cytotoxicity (ADCC) and Antibody-Dependent Cellular Phagocytosis (ADCP).

[0209] The human IgG4 constant region of the monoclonal antibody, antigen-binding fragment thereof, or competing antibody described herein can be modified to prevent Fab arm exchange and/or decrease antibody effector function include, but are not limited to, amino acid modifications F234A/L235A (Alegre et al., 1994); S228P, L235E and S228P/L235E (Reddy et al., 2000).

[0210] As used herein, the term "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.

[0211] The terms "cancer", "cancerous", and "tumor" are not mutually exclusive as used herein. The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by aberrant cell growth/proliferation. Examples of cancers include, but are not limited to, carcinoma, lymphoma (i.e., Hodgkin's and non-Hodgkin's lymphoma), multiple myeloma, blastoma, sarcoma, and leukemia. More particular examples of such cancers include squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastrointestinal cancer, pancreatic cancer, glioma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, hepatic carcinoma, leukemia and other lymphoproliferative disorders, and various types of head and neck cancer.

[0212] The term "susceptible cancer" as used herein refers to a cancer, cells of which express CD47, IRP.alpha., or CD47 and SIRP.alpha. and are responsive to treatment with an antibody or antigen binding fragment thereof, or competing antibody or antigen binding fragment thereof, from the present disclosure that prevent interaction between CD47 and SIRP.alpha..

[0213] The term "autoimmune disease" as used herein refers to when the body's immune system turns against itself and mistakenly attacks healthy cells.

[0214] The term "inflammatory disease" as used herein refers to a disease characterized by inflammation which is a fundamental pathologic process consisting of a dynamic complex of histologically apparent cytologic changes, cellular infiltration, and mediator release that occurs in the affected blood vessels and adjacent tissues in response to an injury or abnormal stimulation caused by a physical, chemical, or biologic agent, including the local reactions and resulting morphologic changes; the destruction or removal of the injurious material; and the responses that lead to repair and healing.

[0215] The term "autoinflammatory disease" as used herein refers to a disease that results when the innate immune system causes inflammation for unknown reasons.

[0216] As used herein, term "treating" or "treat" or "treatment" means slowing, interrupting, arresting, controlling, stopping, reducing, or reversing the progression or severity of a sign, symptom, disorder, condition, or disease, but does not necessarily involve a total elimination of all disease-related signs, symptoms, conditions, or disorders. The term "treating" and the like refer to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition after it has begun to develop.

[0217] As used herein, term "effective amount" refers to the amount or dose of an antibody compound of the present disclosure which, upon single or multiple dose administration to a patient or organ, provides the desired treatment or prevention.

[0218] The precise effective amount for any particular subject will depend upon their size and health, the nature and extent of their condition, and the therapeutics or combination of therapeutics selected for administration. The effective amount for a given patient is determined by routine experimentation and is within the judgment of a clinician. Therapeutically effective amounts of the present antibody compounds can also comprise an amount in the range of from about 0.1 mg/kg to about 150 mg/kg, from about 0.1 mg/kg to about 100 mg/kg, from about 0.1 mg/kg to about 50 mg/kg, or from about 0.05 mg/kg to about 10 mg/kg per single dose administered to a harvested organ or to a patient. Known antibody-based pharmaceuticals provide guidance in this respect. For example, Herceptin.TM. is administered by intravenous infusion of a 21 mg/ml solution, with an initial loading dose of 4 mg/kg body weight and a weekly maintenance dose of 2 mg/kg body weight; Rituxan.TM. is administered weekly at 375 mg/m.sup.2; for example.

[0219] A therapeutically effective amount for any individual patient can be determined by the health care provider by monitoring the effect of the antibody compounds on tumor regression, circulating tumor cells, tumor stem cells or anti-tumor responses. Analysis of the data obtained by these methods permits modification of the treatment regimen during therapy so that optimal amounts of antibody compounds of the present disclosure, whether employed alone or in combination with one another, or in combination with another therapeutic agent, or both, are administered, and so that the duration of treatment can be determined as well. In this way, the dosing/treatment regimen can be modified over the course of therapy so that the lowest amounts of antibody compounds used alone or in combination that exhibit satisfactory efficacy are administered, and so that administration of such compounds is continued only so long as is necessary to successfully treat the patient. Known antibody-based pharmaceuticals provide guidance relating to frequency of administration e.g., whether a pharmaceutical should be delivered daily, weekly, monthly, etc. Frequency and dosage may also depend on the severity of symptoms.

[0220] In some embodiments, antibody compounds of the present disclosure can be used as medicaments in human and veterinary medicine, administered by a variety of routes including, but not limited to, oral, intravenous, intramuscular, intra-arterial, intramedullary, intraperitoneal, intrathecal, intraventricular, transdermal, transcutaneous, topical, subcutaneous, intratumoral, intranasal, enteral, sublingual, intravaginal, intravesicular or rectal routes. The compositions can also be administered directly into a lesion such as a tumor. Dosage treatment may be a single dose schedule or a multiple dose schedule. Hypo sprays may also be used to administer the pharmaceutical compositions. Typically, the therapeutic compositions can be prepared as injectables, either as liquid solutions or suspensions. Solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection can also be prepared. Veterinary applications include the treatment of companion/pet animals, such as cats and dogs; working animals, such as guide or service dogs, and horses; sport animals, such as horses and dogs; zoo animals, such as primates, cats such as lions and tigers, bears, etc.; and other valuable animals kept in captivity.

[0221] Such pharmaceutical compositions can be prepared by methods well known in the art. See, e.g., Remington: The Science and Practice of Pharmacy, 2.sup.st Edition (2005), Lippincott Williams & Wilkins, Philadelphia, Pa., and comprise one or more antibody compounds disclosed herein, and a pharmaceutically acceptable, for example, physiologically acceptable, carrier, diluent, or excipient.

Cancer Indications

[0222] Presently disclosed are anti-SIRP.alpha. mAbs and antigen binding fragments thereof effective as cancer therapeutics which can be administered to patients, preferably parenterally, with susceptible hematologic cancers and solid tumors including, but not limited to, leukemias, including systemic mastocytosis, acute lymphocytic (lymphoblastic) leukemia (ALL), T-cell--ALL, acute myeloid leukemia (AML), myelogenous leukemia, chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CIVIL), myeloproliferative disorder/neoplasm, monocytic cell leukemia, and plasma cell leukemia; multiple myeloma (MM); Waldenstrom's Macroglobulinemia; lymphomas, including histiocytic lymphoma and T-cell lymphoma, B cell lymphomas, including Hodgkin's lymphoma and non-Hodgkin's lymphoma, such as low grade/follicular non-Hodgkin's lymphoma (NHL), cell lymphoma (FCC), mantle cell lymphoma (MCL), diffuse large cell lymphoma (DLCL), small lymphocytic (SL) NHL, intermediate grade/follicular NHL, intermediate grade diffuse NHL, high grade immunoblastic NHL, high grade lymphoblastic NHL, high grade small non-cleaved cell NHL, bulky disease NHL; solid tumors, including ovarian cancer, breast cancer, endometrial cancer, colon cancer (colorectal cancer), rectal cancer, bladder cancer, urothelial cancer, lung cancer (non-small cell lung cancer, adenocarcinoma of the lung, squamous cell carcinoma of the lung), bronchial cancer, bone cancer, prostate cancer, pancreatic cancer, gastric cancer, hepatocellular carcinoma (liver cancer, hepatoma), gall bladder cancer, bile duct cancer, esophageal cancer, renal cell carcinoma, thyroid cancer, squamous cell carcinoma of the head and neck (head and neck cancer), testicular cancer, cancer of the endocrine gland, cancer of the adrenal gland, cancer of the pituitary gland, cancer of the skin, cancer of soft tissues, cancer of blood vessels, cancer of brain, cancer of nerves, cancer of eyes, cancer of meninges, cancer of oropharynx, cancer of hypopharynx, cancer of cervix, and cancer of uterus, glioblastoma, meduloblastoma, astrocytoma, glioma, meningioma, gastrinoma, neuroblastoma, myelodysplastic syndrome, and sarcomas including, but not limited to, osteosarcoma, Ewing's sarcoma, leiomyosarcoma, synovial sarcoma, alveolar soft part sarcoma, angiosarcoma, liposarcoma, fibrosarcoma, rhabdomyosarcoma, and chrondrosarcoma; and melanoma.

Treatment of Cancer

[0223] As is well known to those of ordinary skill in the art, combination therapies are often employed in cancer treatment as single-agent therapies or procedures may not be sufficient to treat or cure the disease or condition. Conventional cancer treatments often involve surgery, radiation treatment, a combination of cytotoxic drugs to achieve additive or synergistic effects, or combinations of any or all of these approaches. Especially useful chemotherapeutic and biologic therapy combinations employ drugs that work via different mechanisms of action, increasing cancer cell control or killing, increasing the ability of the immune system to control cancer cell growth, reducing the likelihood of drug resistance during therapy, and minimizing possible overlapping toxicities by permitting the use of reduced doses of individual drugs.

[0224] Classes of conventional anti-tumor and anti-neoplastic agents useful in the combination therapies encompassed by the present methods are disclosed in Goodman & Gilman's The Pharmacological Basis of Therapeutics, Twelfth Edition (2010) L. L. Brunton, B. A. Chabner, and B. C. Knollmann Eds., Section VIII, "Chemotherapy of Neoplastic Diseases", Chapters 60-63, pp. 1665-1770, McGraw-Hill, NY, include but are not limited to anthracyclines, platinums, taxols, topisomerase inhibitors, anti-metabolites, anti-tumor antibiotics, mitotic inhibitors, and alkylating agents.

[0225] In addition to the foregoing, the methods of the present disclosure are related to treatment of cancer indications and further comprises treating the patient via surgery, radiation, and/or administering to a patient in need thereof an effective amount of a chemical small molecule or biologic drug including, but not limited to, a peptide, polypeptide, protein, nucleic acid therapeutic, conventionally used or currently being developed, to treat tumorous conditions. This includes antibodies and antigen-binding fragments, other than those disclosed herein, cytokines, antisense oligonucleotides, siRNAs, and miRNAs.

[0226] The therapeutic methods disclosed and claimed herein include the use of the antibodies disclosed herein alone, and/or in combinations with one another, and/or with antigen-binding fragments thereof of the present disclosure that bind to SIRP.alpha., and/or with competing antibodies exhibiting appropriate biological/therapeutic activity, as well, for example, all possible combinations of these antibody compounds to achieve the greatest treatment efficacy.

[0227] In addition, the present therapeutic methods also encompass the use of these antibodies, antigen-binding fragments thereof, competing antibodies, and combinations thereof in further in combination with: (1) one or more anti-tumor therapeutic treatments selected from surgery, radiation, anti-tumor, and anti-neoplastic agents or combinations of any of these, or (2) one or more of anti-tumor biological agents or (3) equivalents of any of the foregoing of (1) or (2) as would be apparent to one of ordinary skill in the art, in appropriate combination(s) to achieve the desired therapeutic treatment effect for the particular indication.

[0228] Antibodies and small molecule drugs that increase the immune response to cancer by modulating co-stimulatory or inhibitory interactions that influence the T-cell response to tumor antigens, including inhibitors of immune checkpoints and modulators of co-stimulatory molecules, are also of particular interest in the context of the combination therapeutic methods encompassed herein and include, but are not limited to, other anti-SIRP.alpha. antibodies. Administration of therapeutic agents that bind to the SIRP.alpha. protein, for example, antibodies or small molecules that bind to SIRP.alpha. and prevent interaction between CD47 and SIRP.alpha., are administered to a patient, causing the clearance of cancer cells via phagocytosis. The therapeutic agent that binds to the SIRP.alpha. protein is combined with a therapeutic agent such as an antibody, a chemical small molecule or biologic drug which is directed against one or more additional cellular targets selected from CD47 (Cluster of Differentiation 47), CD70 (Cluster of Differentiation 70), CD200 (OX-2 membrane glycoprotein, Cluster of Differentiation 200), CD154 (Cluster of Differentiation 154, CD40L, CD40 ligand, Cluster of Differentiation 40 ligand), CD223 (Lymphocyte-activation gene 3, LAG3, Cluster of Differentiation 223), KIR (Killer-cell immunoglobulin-like receptors), GITR (TNFRSF18, glucocorticoid-induced TNFR-related protein, activation-inducible TNFR family receptor, AITR, Tumor necrosis factor receptor superfamily member 18), CD20 (Cluster of Differentiation 20), CD28 (Cluster of Differentiation 28), CD40 (Cluster of Differentiation 40, Bp50, CDW40, TNFRSF5, Tumor necrosis factor receptor superfamily member 5, p50), CD86 (B7-2, Cluster of Differentiation 86), CD160 (Cluster of Differentiation 160, BY55, NK1, NK28), CD258 (LIGHT, Cluster of Differentiation 258, Tumor necrosis factor ligand superfamily member 14, TNFSF14, herpesvirus entry mediator ligand (HVEM-L), CD270 (HVEM, Tumor necrosis factor receptor superfamily member 14, herpesvirus entry mediator, Cluster of Differentiation 270, LIGHTR, HVEA), CD275 (ICOSL, ICOS ligand, Inducible T-cell co-stimulator ligand, Cluster of Differentiation 275), CD276 (B7-H3, B7 homolog 3, Cluster of Differentiation 276), OX40L (0X40 Ligand), B7-H4 (B7 homolog 4, VTCN1, V-set domain-containing T-cell activation inhibitor 1), GITRL (Glucocorticoid-induced tumor necrosis factor receptor-ligand, glucocorticoid-induced TNFR-ligand), 4-1BBL (4-1BB ligand), CD3 (Cluster of Differentiation 3, T3D), CD25 (IL2Ra, Cluster of Differentiation 25, Interleukin-2 Receptor a chain, IL-2 Receptor a chain), CD48 (Cluster of Differentiation 48, B-lymphocyte activation marker, BLAST-1, signaling lymphocytic activation molecule 2, SLAMF2), CD66a (Ceacam-1, Carcinoembryonic antigen-related cell adhesion molecule 1, biliary glycoprotein, BGP, BGP1, BGPI, Cluster of Differentiation 66a), CD80 (B7-1, Cluster of Differentiation 80), CD94 (Cluster of Differentiation 94), NKG2A (Natural killer group 2A, killer cell lectin-like receptor subfamily D member 1, KLRD1), CD96 (Cluster of Differentiation 96, TActILE, T-cell activation increased late expression), CD112 (PVRL2, nectin, Poliovirus receptor-related 2, herpesvirus entry mediator B, HVEB, nectin-2, Cluster of Differentiation 112), CD115 (CSF1R, Colony stimulating factor 1 receptor, macrophage colony-stimulating factor receptor, M-CSFR, Cluster of Differentiation 115), CD205 (DEC-205, LY75, Lymphocyte antigen 75, Cluster of Differentiation 205), CD226 (DNAM1, Cluster of Differentiation 226, DNAX Accessory Molecule-1, PTA1, platelet and T-cell activation antigen 1), CD244 (Cluster of Differentiation 244, Natural killer cell receptor 2B4), CD262 (DRS, TrailR2, TRAIL-R2, Tumor necrosis factor receptor superfamily member 10b, TNFRSF10B, Cluster of Differentiation 262, KILLER, TRICK2, TRICKB, ZTNFR9, TRICK2A, TRICK2B), CD284 (Toll-like Receptor-4, TLR4, Cluster of Differentiation 284), CD288 (Toll-like Receptor-8, TLR8, Cluster of Differentiation 288), Leukemia Inhibitor Factor (LIF), TNFSF15 (Tumor necrosis factor superfamily member 15, Vascular endothelial growth inhibitor, VEGI, TL1A), TDO2 (Tryptophan 2,3-dioxygenase, TPH2, TRPO), IGF-1R (Type 1 Insulin-like Growth Factor), GD2 (Disialoganglioside 2), TMIGD2 (Transmembrane and immunoglobulin domain-containing protein 2), RGMB (RGM domain family, member B), VISTA (V-domain immunoglobulin-containing suppressor of T-cell activation, B7-H5, B7 homolog 5), BTNL2 (Butyrophilin-like protein 2), Btn (Butyrophilin family), TIGIT (T-cell Immunoreceptor with Ig and ITIM domains, Vstm3, WUCAM), Siglecs (Sialic acid binding Ig-like lectins), i.e., SIGLEC-15, Neurophilin, VEGFR (Vascular endothelial growth factor receptor), ILT family (LIRs, immunoglobulin-like transcript family, leukocyte immunoglobulin-like receptors), NKG families (Natural killer group families, C-type lectin transmembrane receptors), MICA (MHC class I polypeptide-related sequence A), TGF.beta. (Transforming growth factor (3), STING pathway (Stimulator of interferon gene pathway), Arginase (Arginine amidinase, canavanase, L-arginase, arginine transamidinase), EGFRvIII (Epidermal growth factor receptor variant III), and HHLA2 (B7-H7, B7y, HERV-H LTR-associating protein 2, B7 homolog 7), inhibitors of PD-1 (Programmed cell death protein 1, PD-1, CD279, Cluster of Differentiation 279), PD-L1 (B7-H1, B7 homolog 1, Programmed death-ligand 1, CD274, cluster of Differentiation 274), PD-L2 (B7-DC, Programmed cell death 1 ligand 2, PDCD1LG2, CD273, Cluster of Differentiation 273), CTLA-4 (Cytotoxic T-lymphocyte-associated protein 4, CD152, Cluster of Differentiation 152), BTLA (B- and T-lymphocyte attenuator, CD272, Cluster of Differentiation 272), Indoleamine 2,3-dioxygenase (IDO, IDO1), TIM3 (HAVCR2, Hepatitis A virus cellular receptor 2, T-cell immunoglobulin mucin-3, KIM-3, Kidney injury molecule 3, TIMD-3, T-cell immunoglobulin mucin-domain 3), A2A adenosine receptor (ADO receptor), CD39 (ectonucleoside triphosphate diphosphohydrolase-1, Cluster of Differentiation 39, ENTPD1), and CD73 (Ecto-5'-nucleotidase, 5'-nucleotidase, 5'-NT, Cluster of Differentiation 73), CD27 (Cluster of Differentiation 27), ICOS (CD278, Cluster of Differentiation 278, Inducible T-cell Co-stimulator), CD137 (4-1BB, Cluster of Differentiation 137, tumor necrosis factor receptor superfamily member 9, TNFRSF9), OX40 (CD134, Cluster of Differentiation 134), TNF SF25 (Tumor necrosis factor receptor superfamily member 25), IL-10 (Interleukin-10, human cytokine synthesis inhibitory factor, CSIF), and Galectins.

[0229] ERBITUX.RTM. (cetuximab, Bristol-Meyers Squibb) is an example of an approved recombinant, human/mouse chimeric monoclonal antibody that binds specifically to the extracellular domain of the human epidermal growth factor receptor (EGFR).

[0230] RITUXAN.RTM. (rituximab, Biogen IDEC/Genentech) is an example of an approved anti-CD20 antibody.

[0231] YERVOY.RTM. (ipilimumab; Bristol-Meyers Squibb) is an example of an approved anti-CTLA-4 antibody.

[0232] KEYTRUDA.RTM. (pembrolizumab; Merck) and OPDIVO.RTM. (nivolumab; Bristol-Meyers Squibb Company) are examples of approved anti-PD-1 antibodies.

[0233] TECENTRIQ.TM. (atezolizumab; Roche) is an example of an approved anti-PD-L1 antibody.

[0234] BAVENCIO.TM. (avelumab; Merck KGaA and Pfizer and Eli Lilly and Company) is an example of an approved anti-PD-L1 antibody.

[0235] IMFINZI.TM. (Durvalumab; Medimmune/AstraZeneca) is an example of an approved anti-PD-L1 antibody.

[0236] The Examples illustrate various embodiments of the present disclosure, but they should not be considered as limiting the disclosure to only these particularly disclosed embodiments.

EXAMPLES

Example 1

TABLE-US-00001 [0237] Amino Acid Sequences Light Chain CDRs LCDR1 LCDR2 LCDR3 SEQ ID NO: 1 RASSGVNYMY SEQ ID NO: 2 YTS1LAP SEQ ID NO: 3 QQFTSSPYT SEQ ID NO: 4 RASQSIGTSIH SEQ ID NO: 5 YGSESIS SEQ ID NO: 6 QQSNTWPLT SEQ ID NO: 7 SASSIIGSDFLH SEQ ID NO: 8 RTSILAS SEQ ID NO: 9 QQGSGLPLT SEQ ID NO: 10 KASQDINSHLS SEQ ID NO: 11 RANRLAD SEQ ID NO: 12 LQYDEFPYT SEQ ID NO: 13 SASSSVSYMY SEQ ID NO: 14 LTSNLAS SEQ ID NO: 15 QQWSGNPFT SEQ ID NO: 16 RASENIYSYLT SEQ ID NO: 17 NAKTLAE SEQ ID NO: 18 QHHYGSPRT SEQ ID NO: 19 SASSSISSNFLH SEQ ID NO: 20 RTSILAS SEQ ID NO: 21 QQGSGLPLT SEQ ID NO: 22 SSVSY SEQ ID NO: 23 DTS SEQ ID NO: 24 QQWSSFPWT SEQ ID NO: 25 EDIYDR SEQ ID NO: 26 GTA SEQ ID NO: 27 QQYWTTPWT SEQ ID NO: 28 SSVNY SEQ ID NO: 29 YTS SEQ ID NO: 30 QQFTSSPFT SEQ ID NO: 31 RANRLAT SEQ ID NO: 32 QQYDEFPYT Heavy Chain CDRs HCDR1 HCDR2 HCDR3 SEQ ID NO: 33 KYWIE SEQ ID NO: 34 ELPGSVITNYNEKFKG SEQ ID NO: 35 WGLYDSDDGVDY SEQ ID NO: 36 GCTMS SEQ ID NO: 37 YISNGGDITYYPDTVKG SEQ ID NO: 38 LDGYYYAMDF SEQ ID NO: 39 SYVMH SEQ ID NO: 40 YINPYNDGPKYNEKFKG SEQ ID NO: 41 WDYFNSASGFAF SEQ ID NO: 42 DYFLN SEQ ID NO: 43 RINPYNGDSFINQNFRD SEQ ID NO: 44 GGYDGYFIAYFDY SEQ ID NO: 45 SYTMH SEQ ID NO: 46 YINPTIGYTEYNQKFKD SEQ ID NO: 47 LVITSVLGRAMDY SEQ ID NO: 48 DYGVN SEQ ID NO: 49 WVNTNTRESTYVEDFKG SEQ ID NO: 50 GAYDAYYYYYGMDY SEQ ID NO: 51 TYVMH SEQ ID NO: 52 YINPNNDGPNYNEKFKG SEQ ID NO: 53 WDSYNSAAGFAY SEQ ID NO: 54 GFTLSTYT SEQ ID NO: 55 ITSGDTYT SEQ ID NO: 56 TRDRPLFH SEQ ID NO: 57 GYTFTDYE SEQ ID NO: 58 IHPGSGGT SEQ ID NO: 59 TRAVSGYYAMDY SEQ ID NO: 60 GYTFSNYL SEQ ID NO: 61 IYPGDNNT SEQ ID NO: 62 AGGTDYDGFAN SEQ ID NO: 63 ARAVSGYYAMDY Murine Light Chain (V.sub.L) Variable Domain Sequences and Human Light Chain (V.sub.L) Variable Domain Sequences SEQ ID NO: 64 ENVLTQSPAIMSASLGEKVTMSCRASSGVNYMYWYQQKSDASPKLLIYYTSILAPGVPARFSGSGSG NSYSLTISSMEGEDAATYYCQQFTSSPYTFGGGTKLEIK SEQ ID NO: 65 DILLTQSPAILSVSPGERVSFSCRASQSIGTSIHWYQQRTNGSPRLLIKYGSESISGIPSRFSGSGSGTDFT LSINSVESEDIADYYCQQSNTWPLTFGDGTKLELK SEQ ID NO: 66 EIVLTQSPTTMAASPGEKITIICSASSIIGSDFLHWYQQRPGFSPKFLIYRTS1LASGVPTRFTGSGSGTSY SLTIGTMEAEDVATYYCQQGSGLPLTFGSGTKLEMK SEQ ID NO: 67 DIKLTQSQSSMYSSLGQRVTITCKASQDINSHLSWFQEKPGKSPKTLIYRANRLADGVPSRFSGSGSGQ DYFLTISSLEYEDVGIYYCLQYDEFPYTFGGGTKLEIK SEQ ID NO: 68 QIVLTQSPALMSASPGEKVTMTCSASSSVSYMYWFQQKPRSSPKPWIYLTSNLASGVPARFSGSGSGT SYSLTISSMEAEDAATYYCQQWSGNPFTFGSGTKLEIK SEQ ID NO: 69 DIQMTQSPASLSASVGETVTITCRASENIYSYLTWYKQKQGKSPQLLVYNAKTLAEGVPSRFSGSGSG TQFSLKINSLQPEDFGSYYCQHHYGSPRTFGGGTKLEIK SEQ ID NO: 70 EIVLTQSPTTMAASPGEKITIICSASSSISSNFLHWYQQKPGFSPRFLIYRTSILASGVPTRFSGSGSGTSY SLTIDTMEAEDVATYYCQQGSGLPLTFGSGTKLEIK SEQ ID NO: 71 QIVLTQSPAIMSASPGEKVTMTCSASSSVSYMYWYQQKPGSSPRLLIYDTSNLASGVPVRFSGSGSGTS YSLTISRMEAEDAATYYCQQWSSFPWTFGGGTKLEIK SEQ ID NO: 72 DIQMTQSSSSFSGSLGDRLTINCKASEDIYDRVAWYQQKPGNAPRLLISGTASLETGVLSRFSGSGSGK DYTLSINGLQAEDVATYYCQQYWTTPWTFGGGTKLEIK SEQ ID NO: 73 ENVLTQSPAIMSASLGEKVTMSCRASSSVNYMYWYQQKSDASPKLWIYYTSKLAPGVPARFSGSGSG NSYSLTISSMEGEDAATYYCQQFTSSPFTFGSGTKLEIK SEQ ID NO: 74 DIQMTQSPSSLSASVGDRVTITCKASQDINSHLSWYQQKPGKAPKLLIYRANRLATGVPSRFSGSGSGT DFTFTISSLQPEDIATYYCLQYDEFPYTFGGGTKLEIK SEQ ID NO: 75 DIQMTQSPSSLSASVGDRVTITCKASQDINSHLSWYQQKPGKAPKLLIYRANRLATGVPSRFSGSGSGT DFTFTISSLEYEDIATYYCLQYDEFPYTFGGGTKLEIK SEQ ID NO: 76 DIQMTQSPSSLSASVGDRVTITCKASQDINSHLSWYQQKPGKAPKLLIYRANRLATGVPSRFSGSGSGT DFTFTISSLQPEDIATYYCQQYDEFPYTFGGGTKLEIK SEQ ID NO: 77 DIKMTQSPSSMYASLGQRVTITCKASQDINSHLSWFQEKPGKSPKTLIYRANRLADGVPSRFSGSGSG QDYFLTISSLEYEDVGIYYCLQYDEFPYTFGGGTKLEIK SEQ ID NO: 78 DIQMTQSPSSLSASVGDRVTITCKASEDIYDRVAWYQQKPGKAPKLLIYGTASLETGVPSRFSGSGSGT DFTFTISSLQPEDIATYYCQQYWTTPWTFGGGTKVEIK SEQ ID NO: 79 DIQMTQSPSSLSASVGDRVTITCKASEDIYDRVAWYQQKPGKAPKLLIYGTASLETGVPSRFSGSGSGT DFTLTISSLQPEDFATYYCQQYWTTPWTFGGGTKVEIK SEQ ID NO: 80 DIQMTQSPSSLSASVGDRVTITCKASEDIYDRVAWYQQKPGKAPKLLIYGTASLETGVLSRFSGSGSG TDFTLTISSLQAEDFATYYCQQYWTTPWTFGGGTKVEIK Murine Heavy Chain (V.sub.H) Variable Domain Sequences and Human Heavy Chain (V.sub.H) Variable Domain Sequences SEQ ID NO: 81 QVQLQQSGAELMKPGASVKISCKATGYSFTKYWIEWVKQRPGHGLEWIGHLPGSVITNYNEKFKGK ATFTADTSSNTVYMQLSSLTSEDSAVYYCTKWGLYDSDDGVDYWGQGTTLTVSS SEQ ID NO: 82 EVKLVESGGGLVQPGGSLKLSCAASGFSFSGCTMSWIRQTPERRLEWVAYISNGGDITYYPDTVKGRF TISRDNAKNSLYLQMSSLKSEDTAMYYCARLDGYYYAMDFWGQGTSVTVSS SEQ ID NO: 83 EVQLQQSGPEVVKPGASVKMSCKASGYTFTSYVMHWVKQKPGQGLEWIGYINPYNDGPKYNEKFKG KATLTSDKSSSTAYMELSSLTSEDSAVYFCARWDYFNSASGFAFWGQGTLVTVSA SEQ ID NO: 84 EVQLQQSGPDLVKPGASVKISCKASGYSFTDYFLNWVKQSHGKSLEWIGRINPYNGDSFINQNFRDKA TLTVDKSSTTAHMDLLSLTSEDSAIYYCGRGGYDGYFIAYFDYWGQGSLVTVSA SEQ ID NO: 85 QVQLQQSAAELARPGASVKMSCKASGYTFTSYTMHWVKQRPGQGLEWIGYINPTIGYTEYNQKFKD KTTLTADKSSSTAYMQLSSLTSEDSAVYYCVRLVITSVLGRAMDYWGQGTSVTVSS SEQ ID NO: 86 QIQLVQSGPELKKPGETVKISCKASGYTFTDYGVNWVKQGPGKDLQWMGWVNTNTRESTYVEDFKG RFAFSLETSASTAYLQINNLKNEDSSTYFCARGAYDAYYYYYGMDYWGQGTSVTVSS SEQ ID NO: 87 EVQLQQSGPELVKPGASVKMSCRASGYTFSTYVMHWIKHRPGQGLEWIGYINPNNDGPNYNEKFKG KATLTSDISSSTAYMELSSLTSEDSAVYFCSRWDSYNSAAGFAYWGHGTLVTVSA SEQ ID NO: 88 EVQLQESGGGLVKPGGSLKLSCAASGFTLSTYTMSWVRQTPEKRLEWVAIITSGDTYTYYPDSVKGRF TISRDNAKNTLYLQMSSLKSEDTGMYYCTRDRPLFHWGQGTTLTVST SEQ ID NO: 89 EVQLQESGAELVRPGASVKLSCKALGYTFTDYEIHWVKETPVYGLEWIGDIHPGSGGTANNQKFKGK ATLTADKSSNTAYMELSSLTSEDSAVYYCTRAVSGYYAMDYWGQGTSVTVSS SEQ ID NO: 90 EVQLQESGAELVRPGTSVKMSCKAAGYTFSNYLIGWIKQRPGHGLEWIGDIYPGDNNTNYNEKFRVK ATLTADTSSNTAYMHLTSLTSEDSAIYYCAGGTDYDGFANWGQGTLVTVSA SEQ ID NO: 91 QVQLVQSGAEVKKPGASVKVSCKASGYSFTDYFLNWVRQAPGQGLEWMGRINPYNGDSFINQNFRD RVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGGYDGYFIAYFDYWGAGTTVTVSS SEQ ID NO: 92 QVQLVQSGAEVKKPGSSVKVSCKASGYSFTDYFLNWVRQAPGQGLEWMGRINPYNGDSFINQNFRD RVTITADKSTSTAYMELSSLRSEDTAVYYCARGGYDGYFIAYFDYWGAGTTVTVSS SEQ ID NO: 93 EVQLVQSGAEVKKPGESLKISCKGSGYSFTDYFLNWVRQNfPGKGLEWMGRINPYNGDSFINQNFRDQ VTISADKSISTAYLQWSSLKASDTAMYYCARGGYDGYFIAYFDYWGAGTTVTVSS SEQ ID NO: 94 QVQLVQSGAEVKKPGASVKVSCKASGYSFTDYFLNWVRQAPGQGLEWMGRINPYNGDSFINQNFRD RVTMTVDTSTSTVYMELSSLRSEDTAVYYCARGGYDGYFIAYFDYWGAGTTVTVSS SEQ ID NO: 95 QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYEIHWVRQAPGQGLEWMGDIHPGSGGTANNQKFK GRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARAVSGYYAMDYWGQGTLVTVSS SEQ ID NO: 96 QVQLVQSGAEVKKPGSSVKVSCKASGYTFTDYEIHWVRQAPGQGLEWMGDIHPGSGGTANNQKFKG RVTITADESTSTAYMELSSLRSEDTAVYYCARAVSGYYAMDYWGQGTLVTVSS SEQ ID NO: 97 QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYEIHWVRQAPGQGLEWMGDIHPGSGGTANNQKFK GRVTMTADTSTSTVYMELSSLRSEDTAVYYCTRAVSGYYAMDYWGQGTLVTVSS Murine Light Chain (LC) Sequences and Human Light Chain (LC) Sequences SEQ ID NO: 98 ENVLTQSPAIIVISASLGEKVTMSCRASSGVNYMYWYQQKSDASPKWYYTSILAPGVPARFSGSGSG NSYSLTISSMEGEDAATYYCQQFTSSPYTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLN NFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTS PIVKSFNRNEC SEQ ID NO: 99 DILLTQSPAILSVSPGERVSFSCRASQSIGTSIHWYQQRTNGSPRLLIKYGSESISGIPSRFSGSGSGTDFT LSINSVESEDIADYYCQQSNTWPLTFGDGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYP KDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKS FNRNEC SEQ ID NO: 100 EIVLTQSPTTMAASPGEKITIICSASSIIGSDFLHWYQQRPGFSPKFLIYRTSILASGVPTRFTGSGSGTSY SLTIGTMEAEDVATYYCQQGSGLPLTFGSGTKLEMKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNF YPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIV KSFNRNEC SEQ ID NO: 101 DIQMTQSPSSLSASVGDRVTITCKASQDINSHLSWYQQKPGKAPKLLIYRANRLATGVPSRFSGSGSG TDFTFTISSLQPEDIATYYCLQYDEFPYTFGGGTKLEKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNN FYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSP VTKSFNRGEC SEQ ID NO: 102 DIQMTQSPSSLSASVGDRVTITCKASQDINSHLSWYQQKPGKAPKLLIYRANRLATGVPSRFSGSGSG TDFTFTISSLEYEDIATYYCLQYDEFPYTFGGGTKLEKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNN FYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSP VTKSFNRGEC SEQ ID NO: 103 DIQMTQSPSSLSASVGDRVTITCKASQDINSHLSWYQQKPGKAPKLLIYRANRLATGVPSRFSGSGSG TDFTFTISSLQPEDIATYYCQQYDEFPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLN NFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS PVTKSFNRGEC SEQ ID NO: 104 DIKMTQSPSSMYASLGQRVTITCKASQDINSHLSWFQEKPGKSPKTLIYRANRLADGVPSRFSGSGSG QDYFLTISSLEYEDVGIYYCLQYDEFPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLN NFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS PVTKSFNRGEC SEQ ID NO: 105 DIQMTQSPSSLSASVGDRVTITCKASEDIYDRVAWYQQKPGKAPKLLIYGTASLETGVPSRFSGSGSG

TDFTFTISSLQPEDIATYYCQQYWTTPWTFGGGTKVEKRTVAAPSVFIFPPSDEQLKSGTASVVCLLN NFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS PVTKSFNRGEC SEQ ID NO: 106 DIQMTQSPSSLSASVGDRVTITCKASEDIYDRVAWYQQKPGKAPKLLIYGTASLETGVPSRFSGSGSG TDFTLTISSLQPEDFATYYCQQYWTTPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLL NNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL SSPVTKSFNRGEC SEQ ID NO: 107 DIQMTQSPSSLSASVGDRVTITCKASEDIYDRVAWYQQKPGKAPKLLIYGTASLETGVLSRFSGSGSG TDFTLTISSLQAEDFATYYCQQYWTTPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLL NNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL SSPVTKSFNRGEC SEQ ID NO: 108 DIQMTQSSSSFSGSLGDRLTINCKASEDIYDRVAWYQQKPGNAPRLLISGTASLETGVLSRFSGSGSG KDYTLSINGLQAEDVATYYCQQYWTTPWTFGGGTKLEKRTVAAPSVFIFPPSDEQLKSGTASVVCL LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC Murine Heavy Chain (HC) Sequences and Human Heavy Chain (HC) Sequences SEQ ID QVQLQQSGAELMKPGASVKISCKATGYSFTKYWIEWVKQRPGHGLEWIGEILPGSVITNYNEKFKGK NO: 109 ATFTADTSSNTVYMQLSSLTSEDSAVYYCTKWGLYDSDDGVDYWGQGTTLTVSSAKTTPPSVYPLA PGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETV TCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEV QFSWFVDDVEVHTAQTQPREEQFNSTERSVSELPIMHQDWLNGKEEKCRVNSAAFPAPIEKTISKTKG RPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYS KLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK SEQ ID EVKLVESGGGLVQPGGSLKLSCAASGFSFSGCTMSWIRQTPERRLEWVAYISNGGDITYYPDTVKGR- F NO: 110 TISRDNAKNSLYLQMSSLKSEDTAMYYCARLDGYYYAMDFWGQGTSVTVSSAKTTPPSVYPLAPGS AAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCN VAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFS WFVDDVEVHTAQTQPREEQFNSTERSVSELPIMHQDWLNGKEEKCRVNSAAFPAPIEKTISKTKGRPK APQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLN VQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK SEQ ID EVQLQQSGPEVVKPGASVKMSCKASGYTFTSYVMHWVKQKPGQGLEWIGYINPYNDGPKYNEKEK NO: 111 GKATLTSDKSSSTAYMELSSLTSEDSAVYFCARWDYFNSASGFAFWGQGTLVTVSAAKTTPPSVYP- L APGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSET VTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPE VQFSWFVDDVEVHTAQTQPREEQFNSTERSVSELPIMHQDWLNGKEEKCRVNSAAFPAPIEKTISKTK GRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVY SKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK SEQ ID QVQLVQSGAEVKKPGASVKVSCKASGYSFTDYFLNWVRQAPGQGLEWMGRINPYNGDSFINQNFRD NO: 112 RVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGGYDGYFIAYFDYWGAGTTVTVSSASTKGPSVFP- L APCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKT YTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSQ EDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEK TISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG SFELYSRLTVDKSRWQEGNVESCSVMHEALHNHYTQKSLSLSLGK SEQ ID QVQLVQSGAEVKKPGSSVKVSCKASGYSFTDYFLNWVRQAPGQGLEWMGRINPYNGDSFINQNFRD NO: 113 RVTITADKSTSTAYMELSSLRSEDTAVYYCARGGYDGYFIAYFDYWGAGTTVTVSSASTKGPSVFP- L APCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKT YTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQ EDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEK TISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG SFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK EVQLVQSGAEVKKPGESLKISCKGSGYSFTDYFLNWVRQ1VfPGKGLEWMGRINPYNGDSFINQNFRD SEQ ID QVTISADKSISTAYLQWSSLKASDTAMYYCARGGYDGYFIAYFDYWGAGTTVTVSSASTKGPSVFPL NO: 114 APCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLG- TKT YTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQ EDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEK TISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG SFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK SEQ ID QVQLVQSGAEVKKPGASVKVSCKASGYSFTDYFLNWVRQAPGQGLEWMGRINPYNGDSFINQNFRD NO: 115 RVTMTVDTSTSTVYMELSSLRSEDTAVYYCARGGYDGYFIAYFDYWGAGTTVTVSSASTKGPSVFP- L APCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKT YTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSQ EDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEK TISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG SFELYSRLTVDKSRWQEGNVESCSVMHEALHNHYTQKSLSLSLGK SEQ ID EVQLQQSGPDLVKPGASVKISCKASGYSFTDYFLNWVKQSHGKSLEWIGRINPYNGDSFINQNFRDK NO: 116 ATLTVDKSSTTAHMDLLSLTSEDSAIYYCGRGGYDGYFIAYFDYWGQGSLVTVSAASTKGPSVFPL- A PCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTY TCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSQE DPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKT ISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS FFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK SEQ ID QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYEIHWVRQAPGQGLEWMGDIHPGSGGTANNQKFK NO: 117 GRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARAVSGYYAMDYWGQGTLVTVSSASTKGPSVFPLA PCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTY TCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSQE DPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKT ISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS FFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK SEQ ID QVQLVQSGAEVKKPGSSVKVSCKASGYTFTDYEIHWVRQAPGQGLEWMGDIHPGSGGTANNQKFK NO: 118 GRVTITADESTSTAYMELSSLRSEDTAVYYCARAVSGYYAMDYWGQGTLVTVSSASTKGPSVFPLA- P CSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYT CNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSQED PEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTI SKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS FFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYEIHWVRQAPGQGLEWMGDIHPGSGGTANNQKFK GRVTMTADTSTSTVYMELSSLRSEDTAVYYCTRAVSGYYAMDYWGQGTLVTVSSASTKGPSVFPLA PCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTY SEQ ID TCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQ- E NO: 119 DPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKT ISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS FFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK EVQLQESGAELVRPGASVKLSCKALGYTFTDYEIHWVKETPVYGLEWIGDIHPGSGGTANNQKFKGK ATLTADKSSNTAYMELSSLTSEDSAVYYCTRAVSGYYAMDYWGQGTSVTVSSASTKGPSVFPLAPCS RSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTC SEQ ID NVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQED- P NO: 120 EVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTIS KAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK SIRP.alpha. and SIRP.gamma. Sequences SEQ ID SIRP.alpha. EEELQVIQPDKSVSVAAGESAILHCTVTSLIPVGPIQWFRGAGPARELIYNQKEGHFPRVTTVS NO: 121 ESTKRENMDFSISISNITPADAGTYYCVKFRKGSPDTEFKSGAGTELSVRAKPSAPVVSGPAAR ATPQHTVSFTCESHGFSPRDITLKWFKNGNELSDFQTNVDPVGESVSYSIHSTAKVVLTREDV HSQVICEVAHVTLQGDPLRGTANLSETIRVPPTLEVTQQPVRAENQVNVTCQVRKFYPQRLQ LTWLENGNVSRTETASTVTENKDGTYNWMSWLLVNVSAHRDDVKLTCQVEHDGQPAVSKS HDLKVSAHPKEQGSNTAAENTGSNERNIYIVVGVVCTLLVALLMAALYLVRIRQKKAQGSTS STRLHEPEKNAREITQDTNDITYADLNLPKGKKPAPQAAEPNNHTEYASIQTSPQPASEDTLT YADLDMVHLNRTPKQPAPKPEPSFSEYASVQVPRK SEQ ID EEELQMIQPEKLLLVTVGKTATLHCTVTSLLPVGPVLWFRGVGPGRELIYNQKEGHFPRVTT NO: 122 SIRP.gamma. VSDLTKRNNMDFSIRISSITPADVGTYYCVKFRKGSPENVEFKSGPGTEMALGAKPSAPVVLG PAARTTPEHTVSFTCESHGFSPRDITLKWFKNGNELSDFQTNVDPTGQSVAYSIRSTARVVLD PWDVRSQVICEVAHVTLQGDPLRGTANLSEAIRVPPTLEVTQQPMRVGNQVNVTCQVRKFY PQSLQLTWSENGNVCQRETASTLTENKDGTYNWTSWFLVNISDQRDDVVLTCQVKHDGQL AVSKRLALEVTVHQKDQSSDATPGPASSLTALLLIAVLLGPIYVPWKQKT Human IgG Fc Sequences Human Fc IgG1 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLY SEQ ID NO: 123 SLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFP PKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK Human Fe IgG1- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLY N297Q SLSSVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFP SEQ ID NO: 124 PKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK Human Fc-IgG2 ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLY SEQ ID NO: 125 SLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTV VHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSPGK Human Fc-IgG3 ASTKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLY SEQ ID NO: 126 SLSSVVTVPSSSLGTQTYTCNVNHKPSNTKVDKRVELKTPLGDTTHTCPRCPEPKSCDTPPPCP RCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV VVDVSHEDPEVQFKWYVDGVEVHNAKTKPREEQYNSTFRVVSVLTVLHQDWLNGKEYKC KVSNKALPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESS GQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPG K Human Fc-IgG4 ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLY SEQ ID NO: 127 SLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTV LHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVESCSVMHEAL HNHYTQKSLSLSLG Human Fc-IgG4 ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLY S228P SLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKP SEQ ID NO: 128 KDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTV LHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVESCSVMHEAL HNHYTQKSLSLSLG Human Fc-IgG4 PE ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLY SEQ ID NO: 129 SLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTV LHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVESCSVMHEAL HNHYTQKSLSLSLGK Human Fc-IgG4 PE' ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLY SEQ ID NO: 130 SLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTV LHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVESCSVMHEAL HNHYTQKSLSLSLG Human kappa LC RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSK SEQ ID NO: 131 DSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Example 2

Binding of SIRP Monoclonal Antibodies to SIRP.alpha.

[0238] The binding of anti-SIRP monoclonal antibodies (mAbs) of the present disclosure to SIRP alpha (SIRP.alpha.) was determined by solid phase ELISA using an Fc tagged human SIRP alpha. Binding by soluble anti-SIRP antibodies was measured in vitro.

[0239] Fc tagged human SIRP.alpha. (ACRO #SIG-H5251, genotype variant 1) is adsorbed to high-binding microtiter plates at a concentration of 1 .mu.g/ml diluted in phosphate buffered saline (PBS) overnight at 4.degree. C. The coating solution is removed, the wells are washed and then blocked with 75% casein in PBS containing 0.5% Tween 20 (PBST) for 60 minutes at room temperature while shaking. Blocking solution is removed, the wells are washed and incubated for 60 minutes at room temperature while shaking with either murine or human anti-SIRP mAbs diluted in PBST at a starting concentration of 30 .mu.g/ml and reducing the concentration in 3-fold serial dilutions. Wells are washed three times with PBST and incubated for 60 minutes at room temperature while shaking with an HRP-labeled donkey anti-mouse or anti-human secondary antibody (Jackson ImmunoResearch Laboratories) diluted 1:10,000 in PBST. The wells are washed and then incubated with peroxidase substrate and the absorbance at 450 nm measured. The apparent affinities were calculated using a non-linear fit model (GraphPad Prism).

[0240] As shown in Table 1, all soluble anti-SIRP mAbs bound to human SIRP.alpha. with apparent affinities in the picomolar to nanomolar range. FIG. 1A-FIG. 1V demonstrate representative binding curves for antibodies of the present disclosure.

TABLE-US-00002 TABLE 1 Binding of anti-SIRP Antibodies to human SIRP.alpha.. Human SIRP.alpha. binding K.sub.d (pM) SIRP1 39 SIRP2 182 SIRP3 289 SIRP4 161 SIRP5 65 SIRP6 131 SIRP7 197 SIRP8 57 SIRP9 583 SIRP10 >10,000 SIRP11 194 SIRP12 165 SIRP13 1,565 SIRP14 565 SIRP15 608 SIRP16 >40,000 SIRP17 326 SIRP18 364 SIRP19 >19,000 SIRP20 157 SIRP21 274 SIRP22 >11,000 SIRP23 164

Example 3

Binding of Mouse Anti-SIRP mAbs to THP-1 Cells Expressing SIRP.alpha.

[0241] Binding activity of hybridoma-derived mouse SIRP antibodies SIRP1, SIRP2, and SIRP3 to THP-1 cells which express SIRP.alpha., but not SIRP.gamma., was determined by flow cytometry.

[0242] THP-1 cells were incubated for 60 min at 37.degree. C. with increasing concentrations of the mAbs diluted in PBS, pH 7.2. Cells were then washed with PBS and incubated for an additional hour with Alexa Fluor-647 labeled donkey anti-mouse antibody (Jackson ImmunoResearch Laboratories) in PBS. Cells were washed and binding analyzed using a C6 Accuri Flow Cytometer (Becton Dickinson).

[0243] As shown in FIG. 2, all the antibodies bound to SIRP.alpha. expressing THP-1 cells in a concentration-dependent manner.

Example 4

Binding of SIRP mAbs to SIRP.gamma.

[0244] The binding of anti-SIRP antibodies of the present disclosure to SIRP gamma (SIRP.gamma.) was determined by ELISA using an Fc tagged human SIRP.gamma.. Binding by soluble anti-SIRP antibodies was measured in vitro.

[0245] Fc tagged human SIRP.gamma. (ACRO #SIG-H5253) is adsorbed to high-binding microtiter plates at a concentration of 1 .mu.g/ml in phosphate buffered saline (PBS) overnight at 4.degree. C. The coating solution is removed, the wells are washed and then blocked with 75% casein in PBS containing 0.5% Tween 20 (PBST) for 60 minutes at room temperature while shaking. Blocking solution is removed, the wells are washed and incubated for 60 minutes at room temperature while shaking with anti-SIRP mAbs diluted in PBST at a starting concentration of 30 .mu.g/ml and reducing the concentration in 3-fold serial dilutions. Wells are washed three times with PBST and incubated for 60 minutes at room temperature while shaking with an HRP labeled donkey anti-mouse or anti-human secondary antibody (Jackson ImmonResearch Laboratories) diluted 1:10,000 in PBST. The wells are washed and then incubated with peroxidase substrate and the absorbance at 450 nm determined. The apparent affinities were calculated using a non-linear fit model (GraphPad Prism).

[0246] As shown in Table 2, the soluble anti-SIRP mAbs SIRP2, SIRP3, SIRP4, SIRP5, SIRP6, SIRP7, SIRP9, SIRP10, SIRP11, SIRP12, SIRP16, SIRP17, SIRP18, SIRP20, SIRP21 and SIRP23 bound to human SIRP gamma with apparent affinities in the picomolar or nanomolar range. Additionally, the anti-SIRP mAb SIRP1, SIRP8, SIRP13, SIRP14, SIRP15, SIRP19, and SIRP22 did not appreciably bind human SIRP gamma at mAb concentrations up to 30 .mu.g/ml. FIG. 3A-FIG. 3V demonstrate representative binding curves derived from antibodies of the present disclosure.

TABLE-US-00003 TABLE 2 Binding of anti-SIRP Antibodies to Human SIRP.gamma.. Human SIRP.gamma. binding K.sub.d (pM) SIRP1 *NB SIRP2 734 SIRP3 170 SIRP4 274 SIRP5 126 SIRP6 183 SIRP7 99 SIRP8 *NB SIRP9 510 SIRP10 >10,000 SIRP11 7,223 SIRP12 >12,000 SIRP13 *NB SIRP14 *NB SIRP15 >14,000 SIRP16 *NB SIRP17 >15,000 SIRP18 >34,000 SIRP19 *NB SIRP20 >29,000 SIRP21 >21,000 SIRP22 *NB SIRP23 225 *NB--no binding detected at mAb concentration up to 30 .mu.g/ml

Example 5

Binding of Mouse mAbs to Jurkat T Cells Expressing SIRP.gamma.

[0247] Binding activity of mouse hybridoma-derived SIRP mAbs to Jurkat cells which express SIRP.gamma., but not SIRP.alpha., was determined by flow cytometry.

[0248] Jurkat cells were incubated for 60 min at 37.degree. C., 5% CO.sub.2 with increasing concentrations of the anti-SIRP mAbs diluted in phosphate buffered saline (PBS), pH 7.2. Cells were then washed with PBS and incubated for an additional hour with Alexa Fluor-647 labeled donkey anti-mouse antibody (Jackson ImmunoResearch Laboratories) in PBS. Cells were washed and binding analyzed using a C6 Accuri Flow Cytometer (Becton Dickinson). Alternatively, the cells were incubated for 1 h at 37.degree. C. with the saturating concentration of 10 .mu.g/ml of SIRP mAbs in binding buffer containing 1 mM EDTA (Sigma Aldrich), 1% FBS (Biowest) in PBS (Corning). The cells were then washed and stained for 45 min under the same conditions with donkey anti-mouse IgG fluorescein isothiocyanate (FITC)-linked secondary antibody (Jackson ImmunoResearch Laboratories). The cells were then washed and analyzed by flow cytometry (Attune, Life Technologies).

[0249] As shown in FIG. 4A, SIRP3 bound to SIRP.gamma. expressing Jurkat cells whereas SIRP2 or SIRP1 exhibited no binding. In addition, as shown in FIG. 4B, SIRP9 bound to Jurkat cells at a concentration of 10 .mu.g/ml, comparable to KWAR-23 which has previously been shown to bind to SIRP.gamma. whereas SIRP4 exhibited no binding to SIRP.gamma. on the Jurkat cells.

Example 6

Anti-SIRP mAbs Block CD47/SIRP.alpha. Binding

[0250] To assess the ability of anti-SIRP antibodies of the present disclosure to block the binding of CD47 to SIRP.alpha. in vitro the following method was employed using ELISA plates coated with Histidine (HIS) tagged human SIRP.alpha..

[0251] HIS tagged human SIRP.alpha. (ACRO #SIG-H5225) is adsorbed to high-binding microtiter plates at a concentration of 1 .mu.g/ml diluted in PBS overnight at 4.degree. C. The coating solution is removed, the wells are washed and then blocked with 75% casein in PBS containing 0.5% Tween 20 (PBST) for 60 minutes at room temperature while shaking. Blocking solution is removed, the wells are washed and incubated for 60 minutes at room temperature while shaking with anti-SIRP mAbs diluted in PBST at a starting concentration of 30 .mu.g/ml and reducing the concentration by 3-fold serial dilutions. Wells are washed three times with PBST and incubated for 60 minutes at room temperature while shaking with an FC tagged human CD47 (ACRO #CD7-H5256) at a concentration of 250 ng/ml in PBST. Wells are washed three times with PBST and incubated for 60 minutes at room temperature while shaking with an HRP labeled donkey anti-mouse or anti-human secondary antibody (Jackson ImmunoResearch Laboratories) diluted 1:20,000 in PBST. The wells are washed and then incubated with peroxidase substrate and the absorbance at 450 nm determined. The IC.sub.50 was calculated using a non-linear fit model (GraphPad Prism).

[0252] As shown in Table 3, the soluble anti-SIRP mAbs SIRP2, SIRP3, SIRP4, and SIRP7 block the binding of human SIRP.alpha. to human CD47 with IC.sub.50 values in the nanomolar range. In addition, the soluble anti-SIRP mAbs SIRP1, SIRP5, SIRP6, SIRP8, and SIRP10 were unable to block the binding of human SIRP.alpha. to human CD47 at mAb concentrations of up to 30 FIG. 5A-FIG. 5G demonstrates representative inhibition curves derived from antibodies of the present disclosure.

TABLE-US-00004 TABLE 3 Blocking of CD47/SIRP.alpha. Binding by anti-SIRP Antibodies. SIRP.alpha. Blocking (IC.sub.50 nM) SIRP1 *NB SIRP2 3 SIRP3 2.7 SIRP4 0.71 SIRP5 *NB SIRP6 *NB SIRP7 1.1 SIRP8 *NB SIRP10 *NB *NB--no blocking detected at mAb concentration of up to 30 .mu.g/ml

Example 7

Anti-SIRP Monoclonal Antibodies Block CD47/SIRP.gamma. Binding

[0253] To assess the effect of anti-SIRP mAbs of the present disclosure on binding of CD47 to SIRP.gamma. in vitro the following method was employed using ELISA plates coated with HIS tagged human CD47.

[0254] HIS tagged human CD47 (ACRO #CD7-H5227) is adsorbed to high-binding microtiter plates at a concentration of 2 .mu.g/ml diluted in PBS overnight at 4.degree. C. The coating solution is removed, the wells are washed and then blocked with 75% casein in PBS containing 0.5% Tween 20 (PBST) for 60 minutes at room temperature while shaking. Blocking solution is removed, the wells are washed and incubated for 60 minutes at room temperature while shaking with anti-SIRP mAbs diluted in PBST at a starting concentration of 30 .mu.g/ml and reducing the concentration in 3 fold serial dilutions and 0.5 .mu.g/ml of human SIRP.gamma. (ACRO #SIG-H5253). Wells are washed three times with PBST and incubated for 60 minutes at room temperature while shaking with an HRP labeled donkey anti-mouse or anti-human secondary antibody (Jackson ImmunoResearch Laboratories) diluted 1:20,000 in PBST. The wells are washed and then incubated with peroxidase substrate and the absorbance at 450 nm determined. The IC.sub.50 was calculated using a non-linear fit model (GraphPad Prism).

[0255] As shown in Table 4, the soluble anti-SIRP mAbs SIRP2, SIRP3, SIRP4, SIRP5, SIRP6, and SIRP7 block the binding of human SIRP.gamma. to human CD47 with IC.sub.50 values in the nanomolar range. In addition, the soluble anti-SIRP mAbs SIRP1, SIRP8, SIRP9, and SIRP10 were unable to block the binding of human SIRP.gamma. to human CD47 at mAb concentrations up to 30 .mu.g/ml. FIG. 6A-FIG. 6H demonstrates representative inhibition curves derived from antibodies of the present disclosure.

TABLE-US-00005 TABLE 4 Blocking of CD47/SIRP.gamma. Binding by anti-SIRP Antibodies. SIRP.gamma. Blocking (IC.sub.50 nM) SIRP1 *NB SIRP2 3.5 SIRP3 0.96 SIRP4 0.44 SIRP5 0.163 SIRP6 0.86 SIRP7 0.63 SIRP8 *NB SIRP9 *NB SIRP10 *NB *NB--no blocking detected at mAb concentration up to 30 .mu.g/ml

Example 8

Anti-SIRP mAbs Induce Phagocytosis

[0256] To assess the effect of anti-SIRP mAbs on phagocytosis of tumor cells by macrophages in vitro the following method was employed using flow cytometry.

[0257] Human monocyte-derived macrophages were derived from leukapheresis of healthy human peripheral blood and incubated in AIM-V media (Life Technologies) supplemented with 50 ng/ml M-CSF (Biolegend) for seven days. For the in vitro phagocytosis assay, macrophages were re-plated at a concentration of 3.times.10.sup.4 cells per well in 100 .mu.l of AIM-V media supplemented with 50 ng/ml M-CSF in a 96-well plate and allowed to adhere for 24 hours. Once the effector macrophages adhered to the culture dish, the targeted human cancer cells (Jurkat) were labeled with 1 .mu.M 5(6)-Carboxyfluorescein diacetate N-succinimidyl ester (CFSE; Sigma Aldrich) and added to the macrophage cultures at a concentration of 8.times.10.sup.4 cells in 100 .mu.l of AIM-V media without supplements. Anti-SIRP mAbs were added at various concentrations, FIG. 7A, or 10 .mu.g/ml of the antibodies, FIG. 7B, immediately upon mixture of target and effector cells and allowed to incubate at 37.degree. C. for 3 hours. After 3 hours, all non-phagocytosed cells were removed, and the remaining cells washed three times with PBS. Cells were then incubated in Accutase (Stemcell Technologies) to detach macrophages, collected into microcentrifuge tubes, and incubated in 100 ng of allophycocyanin (APC) labeled CD14 antibodies (BD biosciences) for 30 minutes, washed once, and analyzed by flow cytometry (Attune, Life Technologies) for the percentage of CD14.sup.+ cells that were also CFSE.sup.+, indicating complete phagocytosis.

[0258] As shown in FIG. 7A and FIG. 7B, the soluble anti-SIRP mAbs SIRP4, SIRP9, SIRP11, SIRP12, SIRP13, SIRP14, SIRP15, SIRP16, SIRP17, SIRP18, SIRP19, SIRP20, SIRP21, SIRP22 and SIRP23 induced phagocytosis of Jurkat cells by human macrophages as compared to a murine IgG1 control antibody (Biolegend). In contrast, soluble anti-SIRP mAbs SIRP1, SIRP2, SIRP3, SIRP7, SIRP8 and SIRP10 did not induce the phagocytosis of Jurkat cells by human macrophages.

Example 9

Anti-SIRP mAbs Induce Phagocytosis when Combined with an Anti-CD47 Antibody

[0259] To assess the effect of anti-SIRP mAbs and anti-CD47 mAbs in combination on inducing phagocytosis of tumor cells by macrophages in vitro the following method was employed using flow cytometry.

[0260] Human monocyte-derived macrophages were derived from leukapheresis of healthy human peripheral blood and incubated in AIM-V media (Life Technologies) supplemented with 50 ng/ml M-CSF (Biolegend) for seven days. For the in vitro phagocytosis assay, macrophages were re-plated at a concentration of 3.times.10.sup.4 cells per well in 100 .mu.l of AIM-V media supplemented with 50 ng/ml M-CSF in a 96-well plate and allowed to adhere for 24 hours. Once the effector macrophages adhered to the culture dish, the targeted human cancer cells (Jurkat) were labeled with 1 .mu.M 5(6)-Carboxyfluorescein diacetate N-succinimidyl ester (CFSE; Sigma Aldrich) and added to the macrophage cultures at a concentration of 8.times.10.sup.4 cells in 100 .mu.l of AIM-V media without supplements. Anti-SIRP mAbs alone, an anti-CD47 mAb (known to induce phagocytosis) alone, or anti-SIRP and anti-CD47 mAbs together were added at various concentrations immediately upon mixture of target and effector cells and allowed to incubate at 37.degree. C. for 3 hours. After 3 hours, all non-phagocytosed cells were removed, and the remaining cells washed three times with PBS. Cells were then incubated in Accutase (Stemcell Technologies) to detach macrophages, collected into microcentrifuge tubes, and incubated in 100 ng of allophycocyanin (APC) labeled CD14 antibodies (BD biosciences) for 30 minutes, washed once, and analyzed by flow cytometry (Attune, Life Technologies) for the percentage of CD14.sup.+ cells that were also CFSE.sup.+ indicating complete phagocytosis.

[0261] As shown in FIG. 8A-FIG. 8J, all soluble anti-SIRP mAbs SIRP1, SIRP2, SIRP3, SIRP4, SIRP5, SIRP7, SIRP12, SIRP20, SIRP21 and SIRP22 increase phagocytosis of Jurkat cells by human macrophages to a greater degree when combined with anti-CD47 mAbs compared to either agent alone.

Example 10

Anti-SIRP mAbs Induce Phagocytosis in Combination with Rituxan

[0262] To assess the effect of anti-SIRP mAbs and anti-CD20 mAbs in combination on inducing phagocytosis of tumor cells by macrophages in vitro the following method was employed using flow cytometry.

[0263] Human monocyte-derived macrophages were derived from leukapheresis of healthy human peripheral blood and incubated in AIM-V media (Life Technologies) supplemented with 50 ng/ml M-CSF (Biolegend) for seven days. For the in vitro phagocytosis assay, macrophages were re-plated at a concentration of 3.times.10.sup.4 cells per well in 100 .mu.l of AIM-V media supplemented with 50 ng/ml M-CSF in a 96-well plate and allowed to adhere for 24 hours. Once the effector macrophages adhered to the culture dish, the targeted human cancer cells (RAJI) were labeled with 1 .mu.M 5(6)-Carboxyfluorescein diacetate N-succinimidyl ester (CFSE; Sigma Aldrich) and added to the macrophage cultures at a concentration of 8.times.10.sup.4 cells in 100 .mu.l of AIM-V media without supplements. Anti-SIRP mAbs alone, an anti-CD20 mAb (Rituxan, Roche) alone, or anti-SIRP and anti-CD20 mAbs together were added at various concentrations immediately upon mixture of target and effector cells and allowed to incubate at 37.degree. C. for 3 hours. After 3 hours, all non-phagocytosed cells were removed, and the remaining cells washed three times with PBS. Cells were then incubated in Accutase (Stemcell Technologies) to detach macrophages, collected into microcentrifuge tubes, and incubated in 100 ng of allophycocyanin (APC) labeled CD14 antibodies (BD biosciences) for 30 minutes, washed once, and analyzed by flow cytometry (Attune, Life Technologies) for the percentage of CD14.sup.+ cells that were also CFSE.sup.+ indicating complete phagocytosis.

[0264] As shown in FIG. 9A-FIG. 9D, all soluble anti-SIRP mAbs SIRP1, SIRP2, SIRP3, and SIRP7 increased phagocytosis of RAJI cells by human macrophages to a greater degree when combined with anti-CD20 mAbs compared to either agent alone.

Example 11

Anti-SIRP mAbs Induce Phagocytosis in Combination with Erbitux and Avelumab

[0265] To assess the effect of anti-SIRP mAbs and anti-EGFR mAbs or anti-PD-L1 mAbs in combination on inducing phagocytosis of tumor cells by macrophages in vitro the following method was employed using flow cytometry.

[0266] Human monocyte-derived macrophages were derived from leukapheresis of healthy human peripheral blood and incubated in AIM-V media (Life Technologies) supplemented with 50 ng/ml M-CSF (Biolegend) for seven days. For the in vitro phagocytosis assay, macrophages were re-plated at a concentration of 3.times.10.sup.4 cells per well in 100 .mu.l of AIM-V media supplemented with 50 ng/ml M-CSF in a 96-well plate and allowed to adhere for 24 hours. Once the effector macrophages adhered to the culture dish, the targeted human cancer cells (FaDu or ES-2) were labeled with 1 .mu.M 5(6)-Carboxyfluorescein diacetate N-succinimidyl ester (CF SE; Sigma Aldrich) and added to the macrophage cultures at a concentration of 8.times.10.sup.4 cells in 100 .mu.l of AIM-V media without supplements. Anti-SIRP mAbs alone, an anti-EGFR mAb (Erbitux, Bristol-Myers Squibb) alone, an anti-PD-L1 mAb (Avelumab, Pfizer), or anti-SIRP and anti-EGFR mAbs together were added at various concentrations immediately upon mixture of target and effector cells and allowed to incubate at 37.degree. C. for 3 hours. After 3 hours, all non-phagocytosed cells were removed, and the remaining cells washed three times with PBS. Cells were then incubated in Accutase (Stemcell Technologies) to detach macrophages, collected into microcentrifuge tubes, and incubated in 100 ng of allophycocyanin (APC) labeled CD14 antibodies (BD biosciences) for 30 minutes, washed once, and analyzed by flow cytometry (Attune, Life Technologies) for the percentage of CD14.sup.+ cells that were also CFSE.sup.+ indicating complete phagocytosis.

[0267] As shown in FIG. 10A, soluble anti-SIRP mAb SIRP4 increased phagocytosis of FaDu cells by human macrophages to a greater degree when combined with anti-EGFR mAbs compared to either agent alone. As shown in FIG. 10B, soluble anti-SIRP mAb SIRP4 increased phagocytosis of ES-2 cells by human macrophages to a greater degree when combined with anti-PD-L1 mAbs compared to either agent alone.

Example 12

Anti-SIRP mAbs Bind to Human Macrophages and Dendritic Cells

[0268] To assess the binding of anti-SIRP mAbs to cells expressing SIRP.alpha. such as human macrophages and dendritic cells the following method was employed using flow cytometry.

[0269] Human CD14.sup.+ monocytes, isolated from peripheral blood mononuclear cells (Astarte Biologics) were differentiated in vitro for seven days into macrophages or dendritic cells. For macrophage differentiation, monocytes were incubated in AIM-V media (Life Technologies) supplemented with 50 ng/ml M-CSF (Biolegend) for seven days. For dendritic cell differentiation, monocytes were incubated in AIM-V media (Life Technologies) in the presence of 10% human AB serum (Valley Biomedical), 200 ng/ml GM-CSF (Biolegend) and 50 ng/ml IL-4 (Biolegend). The cells were incubated for 1 h at 37.degree. C., 5% CO.sub.2 with serial dilutions of SIRP mAbs in binding buffer containing 1 mM EDTA (Sigma Aldrich) and 1% FBS (Biowest) in PBS (Corning). The cells were then washed and stained for 45 min under the same conditions with donkey anti-mouse IgG fluorescein isothiocyanate (FITC)-linked secondary antibody (Jackson ImmunoResearch Laboratories). The cells were subsequently stained with anti-CD14 or anti-CD11c conjugated to Alexa Fluor 647 fluorophore (Life Technologies and Biolegend, respectively) for 30 min on ice, washed and analyzed by flow cytometry (Attune, Life Technologies). Binding was assessed as the median FITC fluorescence intensity of CD14.sup.+ or CD11c.sup.+ cells, subtracted from cells stained with the secondary antibody only.

[0270] As shown in Table 5, the soluble anti-SIRP mAbs SIRP3, SIRP4, SIRP5 and SIRP9, as well as OSE-18D5 and KWAR-23, bound to cell-expressed SIRP.alpha. on dendritic cells and/or macrophages with apparent affinities in the picomolar range. FIG. 11 demonstrates representative binding curves derived from the antibodies of the present disclosure.

TABLE-US-00006 TABLE 5 Binding of anti-SIRP mAbs to Human Cells Expressing SIRP.alpha.. Human Human dendritic macrophage cell binding K.sub.d binding K.sub.d (pM) (pM) SIRP3 ND* 3.47 SIRP4 20.7 50 SIRP5 ND* 770 SIRP9 93.7 ND* 18D5 37.3 41.2 KWAR-23 ND* 23.4 *Not Determined

Example 13

Anti-SIRP mAbs Exhibit Variable Binding to Human CD3.sup.+ T Cells

[0271] To assess the binding of anti-SIRP mAbs on human CD3 T cells the following method was employed using flow cytometry.

[0272] Human CD3 T cells, isolated from peripheral blood mononuclear cells (Astarte Biologics) were incubated in 96-well V-bottom plates at 2.5.times.10.sup.5 cells/well for 1 h at 37.degree. C., 5% CO.sub.2 with serial dilutions of SIRP mAbs in binding buffer containing 1 mM EDTA (Sigma Aldrich), 1% FBS (Biowest) in PBS (Corning). The cells were then washed and stained for 45 min under the same conditions with donkey anti-mouse IgG fluorescein isothiocyanate (FITC)-linked secondary antibody (Jackson ImmunoResearch Laboratories). The cells were subsequently stained with anti-CD3 conjugated to 3-carboxy-6,8-difluoro-7-hydroxycoumarin (Pacific Blue) fluorophore (BioLegend) for 30 min on ice, washed and analyzed by flow cytometry (Attune, Life Technologies). Binding was assessed as the median FITC fluorescence intensity of CD3.sup.+ cells, subtracted from CD3.sup.+ cells stained with the secondary antibody only. All SIRP antibodies were generated in-house except for LSB2.20 (BioLegend). For activated T cells, prior to the binding assay CD3 T cells were activated for 72 h in a 96-well flat-bottom plate coated with 10 .mu.g/ml anti-CD3 (clone UCHT1; BioLegend), at 1.times.10.sup.5 cells/well in the presence of 0.5 .mu.g/ml anti-CD28 (clone CD28.2; BioLegend).

[0273] As shown in Table 6, the soluble SIRP3, SIRP7, SIRP9, KWAR-23, and the SIRP.gamma.-specific antibody LSB2.20 bind T cells with affinities in the picomolar range. The affinities of anti-SIRP mAbs SIRP4, SIRP5 and OSE-18D5 are much lower and are in the nanomolar range. FIG. 12A, FIG. 12B, and FIG. 12C demonstrate representative binding curves derived from antibodies of the present disclosure.

TABLE-US-00007 TABLE 6 Binding of anti-SIRP Antibodies to Human T Cells Expressing SIRP.gamma.. Human T cell Human T cell Human T cell binding K.sub.d (pM) binding K.sub.d (pM) binding K.sub.d (pM) Naive Naive Activated SIRP3 80.7 ND ND SIRP4 NC* NC* NC* SIRP5 NC* NC* NC* SIRP7 83.9 ND ND SIRP9 ND 1410 263 18D5 8410 NC* NC* KWAR-23 4.04 1.59 6.22 LSB2.20 750 1260 950 *NC Not calculated; mean fluorescence intensities were comparable to the mIgG1 background level **Not determined.

Example 14

Anti-SIRP mAbs do not Block Soluble CD47/Cellular SIRP.gamma. Binding

[0274] To assess the effect of anti-SIRP antibodies of the present disclosure on blocking the binding of soluble CD47 to cells expressing SIRP.gamma., the following method was employed using soluble human IgG1 Fc tagged human CD47.

[0275] Human T-ALL cells (Jurkat) were incubated at 2.5.times.10.sup.5 cells/well for 1 h at 37.degree. C., CO.sub.2 with 10 .mu.g/ml of anti-SIRP mAbs in binding buffer containing 1 mM EDTA (Sigma Aldrich), 1% FBS (Biowest) in PBS (Corning). Following this, soluble human IgG1 Fc tagged human CD47 (ACRO #CD7-H5256) was added for a final concentration of 50 .mu.g/ml and the cells incubated as previously for another 1 h. The cells were then washed extensively and stained for 45 min under the same conditions with donkey anti-human antibody conjugated to Alexa Fluor 647 (Jackson ImmunoResearch). The samples were analyzed by flow cytometry (Attune, Life Technologies). For analysis, background human IgG1 Fc staining in the absence of soluble Fc tagged CD47 was subtracted from median Alexa Fluor 647 fluorescence intensity. Blocking was assessed as the reduction in background-corrected median fluorescence intensity of Alexa Fluor 647 in the presence of SIRP mAbs compared to murine IgG1 (Biolegend, MOPC-21) control.

[0276] As shown in Table 7, the soluble anti-SIRP mAbs SIRP4, SIRP9, and OSE 18D5 do not block the binding of cell expressed SIRP.gamma. to soluble human CD47. KWAR-23 does block the binding of Jurkat cell expressed SIRP.gamma. to soluble human CD47.

TABLE-US-00008 TABLE 7 Blocking of CD47/SIRP.gamma. Binding by anti-SIRP Antibodies. Blocking of soluble CD47 binding to SIRP.gamma. on Jurkat SIRP4 Non-blocking SIRP9 Non-blocking OSE 18D5 Non-blocking KWAR-23 Blocking

Example 15

Anti-SIRP mAbs Block Soluble CD47/Cellular SIRP.gamma. Binding

[0277] To assess the effect of anti-SIRP antibodies of the present disclosure on binding of soluble CD47 to cells expressing SIRP.gamma., the following method was employed using human macrophages and soluble human IgG1 Fc tagged human CD47.

[0278] Human CD14.sup.+ monocytes, isolated from peripheral blood mononuclear cells (Astarte Biologics) were differentiated in vitro for seven days in AIM-V media (Life Technologies) supplemented with 50 ng/ml M-CSF (Biolegend). Macrophage Fc receptors were then blocked with human Fc receptor blocking solution (Biolegend) for 20 min at room temperature. The cells were then washed and incubated for 1 h at 37T, 5% CO.sub.2 with 10 .mu.g/ml of anti-SIRP mAbs in binding buffer containing 1 mM EDTA (Sigma Aldrich), 1% FBS (Biowest) in PBS (Corning). Following this, soluble human IgG1 Fc tagged human CD47 (ACRO #CD7-H5256) was added for a final concentration of 20 .mu.g/ml and the cells incubated as previously for another 1 h. The cells were then washed extensively and stained for 45 min under the same conditions with donkey anti-human antibody conjugated to Alexa Fluor 647 (Jackson ImmunoResearch). The samples were analyzed by flow cytometry (Attune, Life Technologies). For analysis, background human IgG1 Fc staining in the absence of soluble Fc tagged CD47 was subtracted from median Alexa Fluor 647 fluorescence intensity. Blocking was assessed as the reduction in background-corrected median fluorescence intensity of Alexa Fluor 647 in the presence of SIRP mAbs compared to murine IgG1 (Biolegend, MOPC-21) control. Four different monocyte donors were used in these assays with a minimum of three donors per antibody tested.

[0279] As shown in FIG. 13, the soluble anti-SIRP mAbs SIRP4 and SIRP9 block the binding of cell expressed SIRP.alpha. on macrophages to soluble human CD47. The OSE 18D5 mAb does not block the binding of cell expressed SIRP.alpha. to soluble human CD47.

Example 16

Anti-SIRP mAbs do not Inhibit T Cell Proliferation

[0280] To assess the effect of anti-SIRP mAbs on allogeneic dendritic cell-induced T cell proliferation in vitro the following method was employed using flow cytometry.

[0281] Human monocyte-derived dendritic cells were generated by incubating CD14.sup.+ monocytes (Astarte Biologics) in AIM-V medium (Life Technologies) supplemented with 10% human AB serum (Valley Biomedical), 200 ng/ml GM-CSF (Biolegend) and 50 ng/ml IL-4 (Biolegend) for six days, with addition of fresh, cytokine replete medium on Day 2. For the allogeneic dendritic cell and T cell co-culture assay, immature dendritic cells were re-plated onto a 96-well plate at a concentration of 1.times.10.sup.5 cells per well. CellTrace.TM. Violet (Life Technologies) fluorescent cell proliferation dye-labelled allogeneic healthy donor derived CD3.sup.+ T cells from four different donors (Astarte Biologics) were added to the culture at a 1:5 DC:T cell ratio. Anti-SIRP mAbs were added immediately at the saturating concentration of 10 .mu.g/ml immediately and the cells incubated at 37.degree. C., 5% CO.sub.2 for 6-7 days in a total volume of 200 .mu.l. Cells were then detached by scraping the wells with pipette tips and washed in fluorescence-activated cell sorting buffer (1% FBS, Biowest, in PBS). Cells were then incubated with PerCP-Cy5.5 fluorescent dye labelled CD3 antibody (Biolegend) for 30 minutes on ice, washed once, and analyzed by flow cytometry (Attune, Life Technologies). T cell proliferation was measured by the dilution of the CellTrace.TM. Violet dye within the CD3.sup.+ cell population.

[0282] As shown in FIG. 14A and FIG. 14B, the anti-SIRP mAbs SIRP3, SIRP4, SIRP5, SIRP9, SIRP11, SIRP12, SIRP13, SIRP14, SIRP15, SIRP17, SIRP18, SIRP20, SIRP21, SIRP23 and OSE-18D5 had no significant effect on T cell proliferation compared to control antibody (Biolegend). In contrast, KWAR-23, which blocks both SIRP.alpha. and SIRP.gamma. binding to CD47, inhibited T cell proliferation.

Example 17

Anti-SIRP mAbs do not Inhibit Antigen-Specific T Cell Recall Response

[0283] To assess the effect of anti-SIRP mAbs on antigen recall response in T cells in vitro the following method was employed using flow cytometry.

[0284] Human peripheral blood mononuclear cells from a cytomegalovirus seropositive donor (Astarte Biologics) were labelled with CellTrace.TM. Violet (Life Technologies) fluorescent cell proliferation dye and seeded at 200,000 cells/well in a 96-well plate. The cells were then incubated with different concentrations of cytomegalovirus antigen (Astarte Biologics) in AIM-V medium (Life Technologies) supplemented with 10% human AB serum (Valley Biomedical), which induces an antigen dependent stimulation of T cell proliferation. Anti-SIRP mAbs as well as an anti-CD47 mAb, clone B6H12, (Biolegend) were added immediately at the saturating concentration of 10 .mu.g/ml immediately and the cells incubated at 37.degree. C., 5% CO.sub.2 for five days. T cell proliferation was measured by the dilution of the CellTrace.TM. Violet dye within the CD4.sup.+ cell population.

[0285] As shown in FIG. 15, the soluble anti-SIRP mAbs SIRP4, SIRP5 and SIRP9 did not inhibit the ability of T cells to elicit a CMV antigen recall response. In contrast, the anti-CD47 antibody clone B6H12, which is known to inhibit T cell responses, reduced T cell proliferation compared to murine IgG1 control antibody (Biolegend).

Example 18

TABLE-US-00009 [0286] SIRP.alpha. Antibody Sequences LCDR1 LCDR2 LCDR3 HCDR1 HCDR2 HCDR3 (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID Antibody NO:) NO:) NO:) NO:) NO:) NO:) SIRP1 1 2 3 33 34 35 SIRP2 4 5 6 36 37 38 SIRP3 7 8 9 39 40 41 SIRP4 10 11 12 42 43 44 SIRP5 13 14 15 45 46 47 SIRP6 16 17 18 48 49 50 SIRP7 19 20 21 51 52 53 SIRP8 22 23 24 54 55 56 SIRP9 25 26 27 57 58 59 SIRP10 28 29 30 60 61 62 SIRP11 10 31 12 42 43 44 SIRP12 10 31 12 42 43 44 SIRP13 10 31 32 42 43 44 SIRP14 10 31 12 42 43 44 SIRP15 10 31 12 42 43 44 SIRP16 10 31 32 42 43 44 SIRP17 10 31 12 42 43 44 SIRP18 10 31 12 42 43 44 SIRP19 10 31 32 42 43 44 SIRP20 10 31 12 42 43 44 SIRP21 10 31 12 42 43 44 SIRP22 10 31 32 42 43 44 SIRP23 10 11 12 42 43 44 SIRP24 25 26 27 57 58 63 SIRP25 25 26 27 57 58 63 SIRP26 25 26 27 57 58 63 SIRP27 25 26 27 57 58 63 SIRP28 25 26 27 57 58 63 SIRP29 25 26 27 57 58 63 SIRP30 25 26 27 57 58 59 SIRP31 25 26 27 57 58 59 SIRP32 25 26 27 57 58 59 SIRP33 25 26 27 57 58 63 V.sub.L (SEQ V.sub.H (SEQ LC (SEQ HC (SEQ ID NO:) ID NO:) ID NO:) ID NO:) SIRP1 64 81 98 109 SIRP2 65 82 99 110 SIRP3 66 83 100 111 SIRP4 67 84 SIRP5 68 85 SIRP6 69 86 SIRP7 70 87 SIRP8 71 88 SIRP9 72 89 SIRP10 73 90 SIRP11 74 91 101 112 SIRP12 75 91 102 112 SIRP13 76 91 103 112 SIRP14 74 92 101 113 SIRP15 75 92 102 113 SIRP16 76 92 103 113 SIRP17 74 93 101 114 SIRP18 75 93 102 114 SIRP19 76 93 103 114 SIRP20 74 94 101 115 SIRP21 75 94 102 115 SIRP22 76 94 103 115 SIRP23 77 84 104 116 SIRP24 78 95 105 117 SIRP25 79 95 106 117 SIRP26 80 95 107 117 SIRP27 78 96 105 118 SIRP28 79 96 106 118 SIRP29 80 96 107 118 SIRP30 78 97 105 119 SIRP31 79 97 106 119 SIRP32 80 97 107 119 SIRP33 72 89 108 120

Sequence CWU 1

1

131110PRTArtificial SequenceLCDR1 1Arg Ala Ser Ser Gly Val Asn Tyr Met Tyr1 5 1027PRTArtificial SequenceLCDR2 2Tyr Thr Ser Ile Leu Ala Pro1 539PRTArtificial SequenceLCDR3 3Gln Gln Phe Thr Ser Ser Pro Tyr Thr1 5411PRTArtificial SequenceLCDR1 4Arg Ala Ser Gln Ser Ile Gly Thr Ser Ile His1 5 1057PRTArtificial SequenceLCDR2 5Tyr Gly Ser Glu Ser Ile Ser1 569PRTArtificial SequenceLCDR3 6Gln Gln Ser Asn Thr Trp Pro Leu Thr1 5712PRTArtificial SequenceLCDR1 7Ser Ala Ser Ser Ile Ile Gly Ser Asp Phe Leu His1 5 1087PRTArtificial SequenceLCDR2 8Arg Thr Ser Ile Leu Ala Ser1 599PRTArtificial SequenceLCDR3 9Gln Gln Gly Ser Gly Leu Pro Leu Thr1 51011PRTArtificial SequenceLCDR1 10Lys Ala Ser Gln Asp Ile Asn Ser His Leu Ser1 5 10117PRTArtificial SequenceLCDR2 11Arg Ala Asn Arg Leu Ala Asp1 5129PRTArtificial SequenceLCDR3 12Leu Gln Tyr Asp Glu Phe Pro Tyr Thr1 51310PRTArtificial SequenceLCDR1 13Ser Ala Ser Ser Ser Val Ser Tyr Met Tyr1 5 10147PRTArtificial SequenceLCDR2 14Leu Thr Ser Asn Leu Ala Ser1 5159PRTArtificial SequenceLCDR3 15Gln Gln Trp Ser Gly Asn Pro Phe Thr1 51611PRTArtificial SequenceLCDR1 16Arg Ala Ser Glu Asn Ile Tyr Ser Tyr Leu Thr1 5 10177PRTArtificial SequenceLCDR2 17Asn Ala Lys Thr Leu Ala Glu1 5189PRTArtificial SequenceLCDR3 18Gln His His Tyr Gly Ser Pro Arg Thr1 51912PRTArtificial SequenceLCDR1 19Ser Ala Ser Ser Ser Ile Ser Ser Asn Phe Leu His1 5 10207PRTArtificial SequenceLCDR2 20Arg Thr Ser Ile Leu Ala Ser1 5219PRTArtificial SequenceLCDR3 21Gln Gln Gly Ser Gly Leu Pro Leu Thr1 5225PRTArtificial SequenceLCDR1 22Ser Ser Val Ser Tyr1 5233PRTArtificial SequenceLCDR2 23Asp Thr Ser1249PRTArtificial SequenceLCDR3 24Gln Gln Trp Ser Ser Phe Pro Trp Thr1 5256PRTArtificial SequenceLCDR1 25Glu Asp Ile Tyr Asp Arg1 5263PRTArtificial SequenceLCDR2 26Gly Thr Ala1279PRTArtificial SequenceLCDR3 27Gln Gln Tyr Trp Thr Thr Pro Trp Thr1 5285PRTArtificial SequenceLCDR1 28Ser Ser Val Asn Tyr1 5293PRTArtificial SequenceLCDR2 29Tyr Thr Ser1309PRTArtificial SequenceLCDR3 30Gln Gln Phe Thr Ser Ser Pro Phe Thr1 5317PRTArtificial SequenceLCDR2 31Arg Ala Asn Arg Leu Ala Thr1 5329PRTArtificial SequenceLCDR3 32Gln Gln Tyr Asp Glu Phe Pro Tyr Thr1 5335PRTArtificial SequenceHCDR1 33Lys Tyr Trp Ile Glu1 53417PRTArtificial SequenceHCDR2 34Glu Ile Leu Pro Gly Ser Val Ile Thr Asn Tyr Asn Glu Lys Phe Lys1 5 10 15Gly3512PRTArtificial SequenceHCDR3 35Trp Gly Leu Tyr Asp Ser Asp Asp Gly Val Asp Tyr1 5 10365PRTArtificial SequenceHCDR1 36Gly Cys Thr Met Ser1 53717PRTArtificial SequenceHCDR2 37Tyr Ile Ser Asn Gly Gly Asp Ile Thr Tyr Tyr Pro Asp Thr Val Lys1 5 10 15Gly3810PRTArtificial SequenceHCDR3 38Leu Asp Gly Tyr Tyr Tyr Ala Met Asp Phe1 5 10395PRTArtificial SequenceHCDR1 39Ser Tyr Val Met His1 54017PRTArtificial SequenceHCDR2 40Tyr Ile Asn Pro Tyr Asn Asp Gly Pro Lys Tyr Asn Glu Lys Phe Lys1 5 10 15Gly4112PRTArtificial SequenceHCDR3 41Trp Asp Tyr Phe Asn Ser Ala Ser Gly Phe Ala Phe1 5 10425PRTArtificial SequenceHCDR1 42Asp Tyr Phe Leu Asn1 54317PRTArtificial SequenceHCDR2 43Arg Ile Asn Pro Tyr Asn Gly Asp Ser Phe Ile Asn Gln Asn Phe Arg1 5 10 15Asp4413PRTArtificial SequenceHCDR3 44Gly Gly Tyr Asp Gly Tyr Phe Ile Ala Tyr Phe Asp Tyr1 5 10455PRTArtificial SequenceHCDR1 45Ser Tyr Thr Met His1 54617PRTArtificial SequenceHCDR2 46Tyr Ile Asn Pro Thr Ile Gly Tyr Thr Glu Tyr Asn Gln Lys Phe Lys1 5 10 15Asp4713PRTArtificial SequenceHCDR3 47Leu Val Ile Thr Ser Val Leu Gly Arg Ala Met Asp Tyr1 5 10485PRTArtificial SequenceHCDR1 48Asp Tyr Gly Val Asn1 54917PRTArtificial SequenceHCDR2 49Trp Val Asn Thr Asn Thr Arg Glu Ser Thr Tyr Val Glu Asp Phe Lys1 5 10 15Gly5014PRTArtificial SequenceHCDR3 50Gly Ala Tyr Asp Ala Tyr Tyr Tyr Tyr Tyr Gly Met Asp Tyr1 5 10515PRTArtificial SequenceHCDR1 51Thr Tyr Val Met His1 55217PRTArtificial SequenceHCDR2 52Tyr Ile Asn Pro Asn Asn Asp Gly Pro Asn Tyr Asn Glu Lys Phe Lys1 5 10 15Gly5312PRTArtificial SequenceHCDR3 53Trp Asp Ser Tyr Asn Ser Ala Ala Gly Phe Ala Tyr1 5 10548PRTArtificial SequenceHCDR1 54Gly Phe Thr Leu Ser Thr Tyr Thr1 5558PRTArtificial SequenceHCDR2 55Ile Thr Ser Gly Asp Thr Tyr Thr1 5568PRTArtificial SequenceHCDR3 56Thr Arg Asp Arg Pro Leu Phe His1 5578PRTArtificial SequenceHCDR1 57Gly Tyr Thr Phe Thr Asp Tyr Glu1 5588PRTArtificial SequenceHCDR2 58Ile His Pro Gly Ser Gly Gly Thr1 55912PRTArtificial SequenceHCDR3 59Thr Arg Ala Val Ser Gly Tyr Tyr Ala Met Asp Tyr1 5 10608PRTArtificial SequenceHCDR1 60Gly Tyr Thr Phe Ser Asn Tyr Leu1 5618PRTArtificial SequenceHCDR2 61Ile Tyr Pro Gly Asp Asn Asn Thr1 56211PRTArtificial SequenceHCDR3 62Ala Gly Gly Thr Asp Tyr Asp Gly Phe Ala Asn1 5 106312PRTArtificial SequenceHCDR3 63Ala Arg Ala Val Ser Gly Tyr Tyr Ala Met Asp Tyr1 5 1064106PRTArtificial SequenceLight Chain (VL) Variable Domain Sequence 64Glu Asn Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Leu Gly1 5 10 15Glu Lys Val Thr Met Ser Cys Arg Ala Ser Ser Gly Val Asn Tyr Met 20 25 30Tyr Trp Tyr Gln Gln Lys Ser Asp Ala Ser Pro Lys Leu Leu Ile Tyr 35 40 45Tyr Thr Ser Ile Leu Ala Pro Gly Val Pro Ala Arg Phe Ser Gly Ser 50 55 60Gly Ser Gly Asn Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Gly Glu65 70 75 80Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Phe Thr Ser Ser Pro Tyr Thr 85 90 95Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 10565107PRTArtificial SequenceLight Chain (VL) Variable Domain Sequence 65Asp Ile Leu Leu Thr Gln Ser Pro Ala Ile Leu Ser Val Ser Pro Gly1 5 10 15Glu Arg Val Ser Phe Ser Cys Arg Ala Ser Gln Ser Ile Gly Thr Ser 20 25 30Ile His Trp Tyr Gln Gln Arg Thr Asn Gly Ser Pro Arg Leu Leu Ile 35 40 45Lys Tyr Gly Ser Glu Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Ser65 70 75 80Glu Asp Ile Ala Asp Tyr Tyr Cys Gln Gln Ser Asn Thr Trp Pro Leu 85 90 95Thr Phe Gly Asp Gly Thr Lys Leu Glu Leu Lys 100 10566108PRTArtificial SequenceLight Chain (VL) Variable Domain Sequence 66Glu Ile Val Leu Thr Gln Ser Pro Thr Thr Met Ala Ala Ser Pro Gly1 5 10 15Glu Lys Ile Thr Ile Ile Cys Ser Ala Ser Ser Ile Ile Gly Ser Asp 20 25 30Phe Leu His Trp Tyr Gln Gln Arg Pro Gly Phe Ser Pro Lys Phe Leu 35 40 45Ile Tyr Arg Thr Ser Ile Leu Ala Ser Gly Val Pro Thr Arg Phe Thr 50 55 60Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Gly Thr Met Glu65 70 75 80Ala Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln Gly Ser Gly Leu Pro 85 90 95Leu Thr Phe Gly Ser Gly Thr Lys Leu Glu Met Lys 100 10567107PRTArtificial SequenceLight Chain (VL) Variable Domain Sequence 67Asp Ile Lys Leu Thr Gln Ser Gln Ser Ser Met Tyr Ser Ser Leu Gly1 5 10 15Gln Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Asn Ser His 20 25 30Leu Ser Trp Phe Gln Glu Lys Pro Gly Lys Ser Pro Lys Thr Leu Ile 35 40 45Tyr Arg Ala Asn Arg Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Gln Asp Tyr Phe Leu Thr Ile Ser Ser Leu Glu Tyr65 70 75 80Glu Asp Val Gly Ile Tyr Tyr Cys Leu Gln Tyr Asp Glu Phe Pro Tyr 85 90 95Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 10568106PRTArtificial SequenceLight Chain (VL) Variable Domain Sequence 68Gln Ile Val Leu Thr Gln Ser Pro Ala Leu Met Ser Ala Ser Pro Gly1 5 10 15Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met 20 25 30Tyr Trp Phe Gln Gln Lys Pro Arg Ser Ser Pro Lys Pro Trp Ile Tyr 35 40 45Leu Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser 50 55 60Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu65 70 75 80Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Gly Asn Pro Phe Thr 85 90 95Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 100 10569107PRTArtificial SequenceLight Chain (VL) Variable Domain Sequence 69Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser Val Gly1 5 10 15Glu Thr Val Thr Ile Thr Cys Arg Ala Ser Glu Asn Ile Tyr Ser Tyr 20 25 30Leu Thr Trp Tyr Lys Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Val 35 40 45Tyr Asn Ala Lys Thr Leu Ala Glu Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Gln Phe Ser Leu Lys Ile Asn Ser Leu Gln Pro65 70 75 80Glu Asp Phe Gly Ser Tyr Tyr Cys Gln His His Tyr Gly Ser Pro Arg 85 90 95Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 10570108PRTArtificial SequenceLight Chain (VL) Variable Domain Sequence 70Glu Ile Val Leu Thr Gln Ser Pro Thr Thr Met Ala Ala Ser Pro Gly1 5 10 15Glu Lys Ile Thr Ile Ile Cys Ser Ala Ser Ser Ser Ile Ser Ser Asn 20 25 30Phe Leu His Trp Tyr Gln Gln Lys Pro Gly Phe Ser Pro Arg Phe Leu 35 40 45Ile Tyr Arg Thr Ser Ile Leu Ala Ser Gly Val Pro Thr Arg Phe Ser 50 55 60Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Asp Thr Met Glu65 70 75 80Ala Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln Gly Ser Gly Leu Pro 85 90 95Leu Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 100 10571106PRTArtificial SequenceLight Chain (VL) Variable Domain Sequence 71Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly1 5 10 15Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met 20 25 30Tyr Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro Arg Leu Leu Ile Tyr 35 40 45Asp Thr Ser Asn Leu Ala Ser Gly Val Pro Val Arg Phe Ser Gly Ser 50 55 60Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Met Glu Ala Glu65 70 75 80Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Phe Pro Trp Thr 85 90 95Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 10572107PRTArtificial SequenceLight Chain (VL) Variable Domain Sequence 72Asp Ile Gln Met Thr Gln Ser Ser Ser Ser Phe Ser Gly Ser Leu Gly1 5 10 15Asp Arg Leu Thr Ile Asn Cys Lys Ala Ser Glu Asp Ile Tyr Asp Arg 20 25 30Val Ala Trp Tyr Gln Gln Lys Pro Gly Asn Ala Pro Arg Leu Leu Ile 35 40 45Ser Gly Thr Ala Ser Leu Glu Thr Gly Val Leu Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Lys Asp Tyr Thr Leu Ser Ile Asn Gly Leu Gln Ala65 70 75 80Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln Tyr Trp Thr Thr Pro Trp 85 90 95Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 10573106PRTArtificial SequenceLight Chain (VL) Variable Domain Sequence 73Glu Asn Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Leu Gly1 5 10 15Glu Lys Val Thr Met Ser Cys Arg Ala Ser Ser Ser Val Asn Tyr Met 20 25 30Tyr Trp Tyr Gln Gln Lys Ser Asp Ala Ser Pro Lys Leu Trp Ile Tyr 35 40 45Tyr Thr Ser Lys Leu Ala Pro Gly Val Pro Ala Arg Phe Ser Gly Ser 50 55 60Gly Ser Gly Asn Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Gly Glu65 70 75 80Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Phe Thr Ser Ser Pro Phe Thr 85 90 95Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 100 10574107PRTArtificial SequenceLight Chain (VL) Variable Domain Sequences 74Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Asn Ser His 20 25 30Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Arg Ala Asn Arg Leu Ala Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Ile Ala Thr Tyr Tyr Cys Leu Gln Tyr Asp Glu Phe Pro Tyr 85 90 95Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 10575107PRTArtificial SequenceLight Chain (VL) Variable Domain Sequence 75Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Asn Ser His 20 25 30Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Arg Ala Asn Arg Leu Ala Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Glu Tyr65 70 75 80Glu Asp Ile Ala Thr Tyr Tyr Cys Leu Gln Tyr Asp Glu Phe Pro Tyr 85 90 95Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 10576107PRTArtificial SequenceLight Chain (VL) Variable Domain Sequence 76Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Asn Ser His 20 25 30Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Arg Ala Asn Arg Leu Ala Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr Asp Glu Phe Pro Tyr 85 90 95Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 10577107PRTArtificial SequenceLight Chain (VL) Variable Domain Sequence 77Asp Ile Lys Met Thr Gln Ser Pro Ser Ser Met Tyr Ala Ser Leu Gly1 5 10 15Gln Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Asn Ser His 20 25 30Leu Ser Trp Phe Gln Glu Lys Pro Gly Lys Ser Pro Lys Thr Leu Ile 35 40 45Tyr Arg Ala Asn Arg Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Gln Asp Tyr Phe Leu Thr Ile Ser Ser Leu Glu Tyr65 70 75 80Glu Asp Val Gly Ile Tyr Tyr Cys Leu Gln Tyr Asp Glu Phe Pro Tyr 85 90 95Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 10578107PRTArtificial SequenceLight Chain (VL) Variable Domain Sequence 78Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Glu Asp Ile Tyr Asp Arg 20 25 30Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Gly Thr Ala Ser Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr Trp Thr Thr Pro Trp 85 90 95Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 10579107PRTArtificial SequenceLight Chain (VL) Variable Domain Sequence 79Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Glu Asp Ile Tyr Asp Arg 20 25 30Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Gly Thr Ala Ser Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Trp Thr Thr Pro Trp 85 90 95Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 10580107PRTArtificial SequenceLight Chain (VL) Variable Domain Sequence 80Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Glu Asp Ile Tyr Asp Arg 20 25 30Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Gly Thr Ala Ser Leu Glu Thr Gly Val Leu Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Trp Thr Thr Pro Trp 85 90 95Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 10581121PRTArtificial SequenceHeavy Chain (VH) Variable Domain Sequence 81Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Met Lys Pro Gly Ala1 5 10 15Ser Val Lys Ile Ser Cys Lys Ala Thr Gly Tyr Ser Phe Thr Lys Tyr 20 25 30Trp Ile Glu Trp Val Lys Gln Arg Pro Gly His Gly Leu Glu Trp Ile 35 40 45Gly Glu Ile Leu Pro Gly Ser Val Ile Thr Asn Tyr Asn Glu Lys Phe 50 55 60Lys Gly Lys Ala Thr Phe Thr Ala Asp Thr Ser Ser Asn Thr Val Tyr65 70 75 80Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95Thr Lys Trp Gly Leu Tyr Asp Ser Asp Asp Gly Val Asp Tyr Trp Gly 100 105 110Gln Gly Thr Thr Leu Thr Val Ser Ser 115 12082119PRTArtificial SequenceHeavy Chain (VH) Variable Domain Sequence 82Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Ser Phe Ser Gly Cys 20 25 30Thr Met Ser Trp Ile Arg Gln Thr Pro Glu Arg Arg Leu Glu Trp Val 35 40 45Ala Tyr Ile Ser Asn Gly Gly Asp Ile Thr Tyr Tyr Pro Asp Thr Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75 80Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95Ala Arg Leu Asp Gly Tyr Tyr Tyr Ala Met Asp Phe Trp Gly Gln Gly 100 105 110Thr Ser Val Thr Val Ser Ser 11583121PRTArtificial SequenceHeavy Chain (VH) Variable Domain Sequence 83Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Val Val Lys Pro Gly Ala1 5 10 15Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Val Met His Trp Val Lys Gln Lys Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Pro Lys Tyr Asn Glu Lys Phe 50 55 60Lys Gly Lys Ala Thr Leu Thr Ser Asp Lys Ser Ser Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys 85 90 95Ala Arg Trp Asp Tyr Phe Asn Ser Ala Ser Gly Phe Ala Phe Trp Gly 100 105 110Gln Gly Thr Leu Val Thr Val Ser Ala 115 12084122PRTArtificial SequenceHeavy Chain (VH) Variable Domain Sequence 84Glu Val Gln Leu Gln Gln Ser Gly Pro Asp Leu Val Lys Pro Gly Ala1 5 10 15Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asp Tyr 20 25 30Phe Leu Asn Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile 35 40 45Gly Arg Ile Asn Pro Tyr Asn Gly Asp Ser Phe Ile Asn Gln Asn Phe 50 55 60Arg Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Thr Thr Ala His65 70 75 80Met Asp Leu Leu Ser Leu Thr Ser Glu Asp Ser Ala Ile Tyr Tyr Cys 85 90 95Gly Arg Gly Gly Tyr Asp Gly Tyr Phe Ile Ala Tyr Phe Asp Tyr Trp 100 105 110Gly Gln Gly Ser Leu Val Thr Val Ser Ala 115 12085122PRTArtificial SequenceHeavy Chain (VH) Variable Domain Sequence 85Gln Val Gln Leu Gln Gln Ser Ala Ala Glu Leu Ala Arg Pro Gly Ala1 5 10 15Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45Gly Tyr Ile Asn Pro Thr Ile Gly Tyr Thr Glu Tyr Asn Gln Lys Phe 50 55 60Lys Asp Lys Thr Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr65 70 75 80Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95Val Arg Leu Val Ile Thr Ser Val Leu Gly Arg Ala Met Asp Tyr Trp 100 105 110Gly Gln Gly Thr Ser Val Thr Val Ser Ser 115 12086123PRTArtificial SequenceHeavy Chain (VH) Variable Domain Sequence 86Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu1 5 10 15Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30Gly Val Asn Trp Val Lys Gln Gly Pro Gly Lys Asp Leu Gln Trp Met 35 40 45Gly Trp Val Asn Thr Asn Thr Arg Glu Ser Thr Tyr Val Glu Asp Phe 50 55 60Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr65 70 75 80Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Ser Ser Thr Tyr Phe Cys 85 90 95Ala Arg Gly Ala Tyr Asp Ala Tyr Tyr Tyr Tyr Tyr Gly Met Asp Tyr 100 105 110Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser 115 12087121PRTArtificial SequenceHeavy Chain (VH) Variable Domain Sequence 87Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala1 5 10 15Ser Val Lys Met Ser Cys Arg Ala Ser Gly Tyr Thr Phe Ser Thr Tyr 20 25 30Val Met His Trp Ile Lys His Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45Gly Tyr Ile Asn Pro Asn Asn Asp Gly Pro Asn Tyr Asn Glu Lys Phe 50 55 60Lys Gly Lys Ala Thr Leu Thr Ser Asp Ile Ser Ser Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys 85 90 95Ser Arg Trp Asp Ser Tyr Asn Ser Ala Ala Gly Phe Ala Tyr Trp Gly 100 105 110His Gly Thr Leu Val Thr Val Ser Ala 115 12088115PRTArtificial SequenceHeavy Chain (VH) Variable Domain Sequence 88Glu Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly1 5 10 15Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Ser Thr Tyr 20 25 30Thr Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val 35 40 45Ala Ile Ile Thr Ser Gly Asp Thr Tyr Thr Tyr Tyr Pro Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Gly Met Tyr Tyr Cys 85 90 95Thr Arg Asp Arg Pro Leu Phe His Trp Gly Gln Gly Thr Thr Leu Thr 100 105 110Val Ser Thr 11589119PRTArtificial SequenceHeavy Chain (VH) Variable Domain Sequence 89Glu Val Gln Leu Gln Glu Ser Gly Ala Glu Leu Val Arg Pro Gly Ala1 5 10 15Ser Val Lys Leu Ser Cys Lys Ala Leu Gly Tyr Thr Phe Thr Asp Tyr 20 25 30Glu Ile His Trp Val Lys Glu Thr Pro Val Tyr Gly Leu Glu Trp Ile 35 40 45Gly Asp Ile His Pro Gly Ser Gly Gly Thr Ala Asn Asn Gln Lys Phe 50 55 60Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Asn Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95Thr Arg Ala Val Ser Gly Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110Thr Ser Val Thr Val Ser Ser 11590118PRTArtificial SequenceHeavy Chain (VH) Variable Domain Sequence 90Glu Val Gln Leu Gln Glu Ser Gly Ala Glu Leu Val Arg Pro Gly Thr1 5 10 15Ser Val Lys Met Ser Cys Lys Ala Ala Gly Tyr Thr Phe Ser Asn Tyr 20 25 30Leu Ile Gly Trp Ile Lys Gln Arg Pro Gly His Gly Leu Glu Trp Ile 35 40 45Gly Asp Ile Tyr Pro Gly Asp Asn Asn Thr Asn Tyr Asn Glu Lys Phe 50 55 60Arg Val Lys Ala Thr Leu Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr65 70 75 80Met His Leu Thr Ser Leu Thr Ser Glu Asp Ser Ala Ile Tyr Tyr Cys 85 90 95Ala Gly Gly Thr Asp Tyr Asp Gly Phe Ala Asn Trp Gly Gln Gly Thr 100 105 110Leu Val Thr Val Ser Ala 11591122PRTArtificial SequenceHeavy Chain (VH) Variable Domain Sequence 91Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asp Tyr 20 25 30Phe Leu Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Arg Ile Asn Pro Tyr Asn Gly Asp Ser Phe Ile Asn Gln Asn Phe 50 55 60Arg Asp Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Gly Tyr Asp Gly Tyr Phe Ile Ala Tyr Phe Asp Tyr Trp 100 105 110Gly Ala Gly Thr Thr Val Thr Val Ser Ser 115 12092122PRTArtificial SequenceHeavy Chain (VH) Variable Domain Sequence 92Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asp Tyr 20 25 30Phe Leu Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Arg Ile Asn Pro Tyr Asn Gly Asp Ser Phe Ile Asn Gln Asn Phe 50 55 60Arg Asp Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Gly Tyr Asp Gly Tyr Phe Ile Ala Tyr Phe Asp Tyr Trp 100 105 110Gly Ala Gly Thr Thr Val Thr Val Ser Ser 115 12093122PRTArtificial SequenceHeavy Chain (VH) Variable Domain Sequence 93Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu1 5 10 15Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Asp Tyr 20 25 30Phe Leu Asn Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met 35 40 45Gly Arg Ile Asn Pro Tyr Asn Gly Asp Ser Phe Ile Asn Gln Asn Phe 50 55 60Arg Asp Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr65 70 75 80Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys 85 90 95Ala Arg Gly Gly Tyr Asp Gly Tyr Phe Ile Ala Tyr Phe Asp Tyr Trp 100 105 110Gly Ala Gly Thr Thr Val Thr Val Ser Ser 115 12094122PRTArtificial SequenceHeavy Chain (VH) Variable Domain Sequence 94Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asp Tyr 20 25 30Phe Leu Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Arg Ile Asn Pro Tyr Asn Gly Asp Ser Phe Ile Asn Gln Asn Phe 50 55 60Arg Asp Arg Val Thr Met Thr Val Asp Thr Ser Thr Ser Thr Val Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Gly Tyr Asp Gly Tyr Phe Ile Ala Tyr Phe Asp Tyr Trp 100 105 110Gly Ala Gly Thr Thr Val Thr Val Ser Ser 115 12095119PRTArtificial SequenceHeavy Chain (VH) Variable Domain Sequence 95Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30Glu Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Asp Ile His Pro Gly Ser Gly Gly Thr Ala Asn Asn Gln Lys Phe 50 55 60Lys Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Ala Val Ser Gly Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val Ser Ser 11596119PRTArtificial SequenceHeavy Chain (VH) Variable Domain Sequence 96Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30Glu Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Asp Ile His Pro Gly Ser Gly Gly Thr Ala Asn Asn Gln Lys Phe 50 55 60Lys Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Ala Val Ser Gly Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val Ser Ser 11597119PRTArtificial SequenceHeavy Chain (VH) Variable Domain Sequence 97Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30Glu Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu

Glu Trp Met 35 40 45Gly Asp Ile His Pro Gly Ser Gly Gly Thr Ala Asn Asn Gln Lys Phe 50 55 60Lys Gly Arg Val Thr Met Thr Ala Asp Thr Ser Thr Ser Thr Val Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Thr Arg Ala Val Ser Gly Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val Ser Ser 11598213PRTArtificial SequenceLight Chain (LC) Sequence 98Glu Asn Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Leu Gly1 5 10 15Glu Lys Val Thr Met Ser Cys Arg Ala Ser Ser Gly Val Asn Tyr Met 20 25 30Tyr Trp Tyr Gln Gln Lys Ser Asp Ala Ser Pro Lys Leu Leu Ile Tyr 35 40 45Tyr Thr Ser Ile Leu Ala Pro Gly Val Pro Ala Arg Phe Ser Gly Ser 50 55 60Gly Ser Gly Asn Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Gly Glu65 70 75 80Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Phe Thr Ser Ser Pro Tyr Thr 85 90 95Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala Asp Ala Ala Pro 100 105 110Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu Thr Ser Gly Gly 115 120 125Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile Asn 130 135 140Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val Leu Asn145 150 155 160Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser Ser 165 170 175Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr Thr 180 185 190Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys Ser Phe 195 200 205Asn Arg Asn Glu Cys 21099214PRTArtificial SequenceLight Chain (LC) Sequence 99Asp Ile Leu Leu Thr Gln Ser Pro Ala Ile Leu Ser Val Ser Pro Gly1 5 10 15Glu Arg Val Ser Phe Ser Cys Arg Ala Ser Gln Ser Ile Gly Thr Ser 20 25 30Ile His Trp Tyr Gln Gln Arg Thr Asn Gly Ser Pro Arg Leu Leu Ile 35 40 45Lys Tyr Gly Ser Glu Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Ser65 70 75 80Glu Asp Ile Ala Asp Tyr Tyr Cys Gln Gln Ser Asn Thr Trp Pro Leu 85 90 95Thr Phe Gly Asp Gly Thr Lys Leu Glu Leu Lys Arg Ala Asp Ala Ala 100 105 110Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu Thr Ser Gly 115 120 125Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile 130 135 140Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val Leu145 150 155 160Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser 165 170 175Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr 180 185 190Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys Ser 195 200 205Phe Asn Arg Asn Glu Cys 210100215PRTArtificial SequenceLight Chain (LC) Sequence 100Glu Ile Val Leu Thr Gln Ser Pro Thr Thr Met Ala Ala Ser Pro Gly1 5 10 15Glu Lys Ile Thr Ile Ile Cys Ser Ala Ser Ser Ile Ile Gly Ser Asp 20 25 30Phe Leu His Trp Tyr Gln Gln Arg Pro Gly Phe Ser Pro Lys Phe Leu 35 40 45Ile Tyr Arg Thr Ser Ile Leu Ala Ser Gly Val Pro Thr Arg Phe Thr 50 55 60Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Gly Thr Met Glu65 70 75 80Ala Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln Gly Ser Gly Leu Pro 85 90 95Leu Thr Phe Gly Ser Gly Thr Lys Leu Glu Met Lys Arg Ala Asp Ala 100 105 110Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu Thr Ser 115 120 125Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp 130 135 140Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val145 150 155 160Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met 165 170 175Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser 180 185 190Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys 195 200 205Ser Phe Asn Arg Asn Glu Cys 210 215101214PRTArtificial SequenceLight Chain (LC) Sequence 101Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Asn Ser His 20 25 30Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Arg Ala Asn Arg Leu Ala Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Ile Ala Thr Tyr Tyr Cys Leu Gln Tyr Asp Glu Phe Pro Tyr 85 90 95Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala 100 105 110Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln145 150 155 160Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205Phe Asn Arg Gly Glu Cys 210102214PRTArtificial SequenceLight Chain (LC) Sequence 102Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Asn Ser His 20 25 30Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Arg Ala Asn Arg Leu Ala Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Glu Tyr65 70 75 80Glu Asp Ile Ala Thr Tyr Tyr Cys Leu Gln Tyr Asp Glu Phe Pro Tyr 85 90 95Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala 100 105 110Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln145 150 155 160Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205Phe Asn Arg Gly Glu Cys 210103214PRTArtificial SequenceLight Chain (LC) Sequence 103Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Asn Ser His 20 25 30Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Arg Ala Asn Arg Leu Ala Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr Asp Glu Phe Pro Tyr 85 90 95Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala 100 105 110Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln145 150 155 160Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205Phe Asn Arg Gly Glu Cys 210104214PRTArtificial SequenceLight Chain (LC) Sequence 104Asp Ile Lys Met Thr Gln Ser Pro Ser Ser Met Tyr Ala Ser Leu Gly1 5 10 15Gln Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Asn Ser His 20 25 30Leu Ser Trp Phe Gln Glu Lys Pro Gly Lys Ser Pro Lys Thr Leu Ile 35 40 45Tyr Arg Ala Asn Arg Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Gln Asp Tyr Phe Leu Thr Ile Ser Ser Leu Glu Tyr65 70 75 80Glu Asp Val Gly Ile Tyr Tyr Cys Leu Gln Tyr Asp Glu Phe Pro Tyr 85 90 95Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala 100 105 110Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln145 150 155 160Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205Phe Asn Arg Gly Glu Cys 210105214PRTArtificial SequenceLight Chain (LC) Sequence 105Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Glu Asp Ile Tyr Asp Arg 20 25 30Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Gly Thr Ala Ser Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr Trp Thr Thr Pro Trp 85 90 95Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln145 150 155 160Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205Phe Asn Arg Gly Glu Cys 210106214PRTArtificial SequenceLight Chain (LC) Sequence 106Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Glu Asp Ile Tyr Asp Arg 20 25 30Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Gly Thr Ala Ser Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Trp Thr Thr Pro Trp 85 90 95Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln145 150 155 160Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205Phe Asn Arg Gly Glu Cys 210107214PRTArtificial SequenceLight Chain (LC) Sequence 107Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Glu Asp Ile Tyr Asp Arg 20 25 30Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Gly Thr Ala Ser Leu Glu Thr Gly Val Leu Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Trp Thr Thr Pro Trp 85 90 95Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln145 150 155 160Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205Phe Asn Arg Gly Glu Cys 210108214PRTArtificial SequenceLight Chain (LC) Sequence 108Asp Ile Gln Met Thr Gln Ser Ser Ser Ser Phe Ser Gly Ser Leu Gly1 5 10 15Asp Arg Leu Thr Ile Asn Cys Lys Ala Ser Glu Asp Ile Tyr Asp Arg 20 25 30Val Ala Trp Tyr Gln Gln Lys Pro Gly Asn Ala Pro Arg Leu Leu Ile 35 40 45Ser Gly Thr Ala Ser Leu Glu Thr Gly Val Leu Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Lys Asp Tyr Thr Leu Ser Ile Asn Gly Leu Gln Ala65 70 75 80Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln Tyr Trp Thr Thr Pro Trp 85 90 95Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala 100 105 110Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln145 150 155 160Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190Ala

Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205Phe Asn Arg Gly Glu Cys 210109445PRTArtificial SequenceHeavy Chain (HC) Sequence 109Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Met Lys Pro Gly Ala1 5 10 15Ser Val Lys Ile Ser Cys Lys Ala Thr Gly Tyr Ser Phe Thr Lys Tyr 20 25 30Trp Ile Glu Trp Val Lys Gln Arg Pro Gly His Gly Leu Glu Trp Ile 35 40 45Gly Glu Ile Leu Pro Gly Ser Val Ile Thr Asn Tyr Asn Glu Lys Phe 50 55 60Lys Gly Lys Ala Thr Phe Thr Ala Asp Thr Ser Ser Asn Thr Val Tyr65 70 75 80Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95Thr Lys Trp Gly Leu Tyr Asp Ser Asp Asp Gly Val Asp Tyr Trp Gly 100 105 110Gln Gly Thr Thr Leu Thr Val Ser Ser Ala Lys Thr Thr Pro Pro Ser 115 120 125Val Tyr Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val 130 135 140Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val145 150 155 160Thr Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala 165 170 175Val Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro 180 185 190Ser Ser Thr Trp Pro Ser Glu Thr Val Thr Cys Asn Val Ala His Pro 195 200 205Ala Ser Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys Gly 210 215 220Cys Lys Pro Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile225 230 235 240Phe Pro Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys 245 250 255Val Thr Cys Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln 260 265 270Phe Ser Trp Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr Gln 275 280 285Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu Leu 290 295 300Pro Ile Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg305 310 315 320Val Asn Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys 325 330 335Thr Lys Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro 340 345 350Lys Glu Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr 355 360 365Asp Phe Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln 370 375 380Pro Ala Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp Gly385 390 395 400Ser Tyr Phe Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp Glu 405 410 415Ala Gly Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn 420 425 430His His Thr Glu Lys Ser Leu Ser His Ser Pro Gly Lys 435 440 445110443PRTArtificial SequenceHeavy Chain (HC) Sequence 110Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Ser Phe Ser Gly Cys 20 25 30Thr Met Ser Trp Ile Arg Gln Thr Pro Glu Arg Arg Leu Glu Trp Val 35 40 45Ala Tyr Ile Ser Asn Gly Gly Asp Ile Thr Tyr Tyr Pro Asp Thr Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75 80Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95Ala Arg Leu Asp Gly Tyr Tyr Tyr Ala Met Asp Phe Trp Gly Gln Gly 100 105 110Thr Ser Val Thr Val Ser Ser Ala Lys Thr Thr Pro Pro Ser Val Tyr 115 120 125Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val Thr Leu 130 135 140Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr Trp145 150 155 160Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro Ser Ser 180 185 190Thr Trp Pro Ser Glu Thr Val Thr Cys Asn Val Ala His Pro Ala Ser 195 200 205Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys Gly Cys Lys 210 215 220Pro Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe Pro225 230 235 240Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val Thr 245 250 255Cys Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser 260 265 270Trp Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr Gln Pro Arg 275 280 285Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro Ile 290 295 300Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val Asn305 310 315 320Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys 325 330 335Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys Glu 340 345 350Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asp Phe 355 360 365Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala 370 375 380Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp Gly Ser Tyr385 390 395 400Phe Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly 405 410 415Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn His His 420 425 430Thr Glu Lys Ser Leu Ser His Ser Pro Gly Lys 435 440111445PRTArtificial SequenceHeavy Chain (HC) Sequence 111Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Val Val Lys Pro Gly Ala1 5 10 15Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Val Met His Trp Val Lys Gln Lys Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Pro Lys Tyr Asn Glu Lys Phe 50 55 60Lys Gly Lys Ala Thr Leu Thr Ser Asp Lys Ser Ser Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys 85 90 95Ala Arg Trp Asp Tyr Phe Asn Ser Ala Ser Gly Phe Ala Phe Trp Gly 100 105 110Gln Gly Thr Leu Val Thr Val Ser Ala Ala Lys Thr Thr Pro Pro Ser 115 120 125Val Tyr Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val 130 135 140Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val145 150 155 160Thr Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala 165 170 175Val Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro 180 185 190Ser Ser Thr Trp Pro Ser Glu Thr Val Thr Cys Asn Val Ala His Pro 195 200 205Ala Ser Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys Gly 210 215 220Cys Lys Pro Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile225 230 235 240Phe Pro Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys 245 250 255Val Thr Cys Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln 260 265 270Phe Ser Trp Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr Gln 275 280 285Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu Leu 290 295 300Pro Ile Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg305 310 315 320Val Asn Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys 325 330 335Thr Lys Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro 340 345 350Lys Glu Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr 355 360 365Asp Phe Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln 370 375 380Pro Ala Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp Gly385 390 395 400Ser Tyr Phe Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp Glu 405 410 415Ala Gly Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn 420 425 430His His Thr Glu Lys Ser Leu Ser His Ser Pro Gly Lys 435 440 445112449PRTArtificial SequenceHeavy Chain (HC) Sequence 112Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asp Tyr 20 25 30Phe Leu Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Arg Ile Asn Pro Tyr Asn Gly Asp Ser Phe Ile Asn Gln Asn Phe 50 55 60Arg Asp Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Gly Tyr Asp Gly Tyr Phe Ile Ala Tyr Phe Asp Tyr Trp 100 105 110Gly Ala Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr 130 135 140Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr145 150 155 160Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp 195 200 205His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr 210 215 220Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro225 230 235 240Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp 260 265 270Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val 290 295 300Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu305 310 315 320Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys 325 330 335Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355 360 365Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu385 390 395 400Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys 405 410 415Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly 435 440 445Lys113449PRTArtificial SequenceHeavy Chain (HC) Sequence 113Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asp Tyr 20 25 30Phe Leu Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Arg Ile Asn Pro Tyr Asn Gly Asp Ser Phe Ile Asn Gln Asn Phe 50 55 60Arg Asp Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Gly Tyr Asp Gly Tyr Phe Ile Ala Tyr Phe Asp Tyr Trp 100 105 110Gly Ala Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr 130 135 140Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr145 150 155 160Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp 195 200 205His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr 210 215 220Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro225 230 235 240Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp 260 265 270Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val 290 295 300Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu305 310 315 320Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys 325 330 335Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355 360 365Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu385 390 395 400Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys 405 410 415Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly 435 440 445Lys114449PRTArtificial SequenceHeavy Chain (HC) Sequence 114Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu1 5 10 15Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Asp Tyr 20 25 30Phe Leu Asn Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met 35 40 45Gly Arg Ile Asn Pro Tyr Asn Gly Asp Ser Phe Ile Asn Gln Asn Phe 50 55 60Arg Asp Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr65 70 75 80Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys 85 90 95Ala Arg Gly Gly Tyr Asp Gly Tyr Phe Ile Ala Tyr Phe Asp Tyr Trp 100 105 110Gly Ala Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr 130 135 140Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr145 150 155 160Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175Ala Val Leu Gln Ser

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp 195 200 205His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr 210 215 220Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro225 230 235 240Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp 260 265 270Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val 290 295 300Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu305 310 315 320Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys 325 330 335Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355 360 365Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu385 390 395 400Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys 405 410 415Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly 435 440 445Lys115449PRTArtificial SequenceHeavy Chain (HC) Sequence 115Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asp Tyr 20 25 30Phe Leu Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Arg Ile Asn Pro Tyr Asn Gly Asp Ser Phe Ile Asn Gln Asn Phe 50 55 60Arg Asp Arg Val Thr Met Thr Val Asp Thr Ser Thr Ser Thr Val Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Gly Tyr Asp Gly Tyr Phe Ile Ala Tyr Phe Asp Tyr Trp 100 105 110Gly Ala Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr 130 135 140Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr145 150 155 160Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp 195 200 205His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr 210 215 220Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro225 230 235 240Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp 260 265 270Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val 290 295 300Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu305 310 315 320Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys 325 330 335Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355 360 365Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu385 390 395 400Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys 405 410 415Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly 435 440 445Lys116449PRTArtificial SequenceHeavy Chain (HC) Sequence 116Glu Val Gln Leu Gln Gln Ser Gly Pro Asp Leu Val Lys Pro Gly Ala1 5 10 15Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asp Tyr 20 25 30Phe Leu Asn Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile 35 40 45Gly Arg Ile Asn Pro Tyr Asn Gly Asp Ser Phe Ile Asn Gln Asn Phe 50 55 60Arg Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Thr Thr Ala His65 70 75 80Met Asp Leu Leu Ser Leu Thr Ser Glu Asp Ser Ala Ile Tyr Tyr Cys 85 90 95Gly Arg Gly Gly Tyr Asp Gly Tyr Phe Ile Ala Tyr Phe Asp Tyr Trp 100 105 110Gly Gln Gly Ser Leu Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro 115 120 125Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr 130 135 140Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr145 150 155 160Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp 195 200 205His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr 210 215 220Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro225 230 235 240Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp 260 265 270Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val 290 295 300Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu305 310 315 320Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys 325 330 335Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355 360 365Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu385 390 395 400Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys 405 410 415Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly 435 440 445Lys117446PRTArtificial SequenceHeavy Chain (HC) Sequence 117Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30Glu Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Asp Ile His Pro Gly Ser Gly Gly Thr Ala Asn Asn Gln Lys Phe 50 55 60Lys Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Ala Val Ser Gly Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu 130 135 140Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp145 150 155 160Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro 195 200 205Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro 210 215 220Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe225 230 235 240Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 245 250 255Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val 260 265 270Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 275 280 285Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val 290 295 300Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys305 310 315 320Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser 325 330 335Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 340 345 350Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 355 360 365Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375 380Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp385 390 395 400Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp 405 410 415Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 420 425 430Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 435 440 445118446PRTArtificial SequenceHeavy Chain (HC) Sequence 118Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30Glu Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Asp Ile His Pro Gly Ser Gly Gly Thr Ala Asn Asn Gln Lys Phe 50 55 60Lys Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Ala Val Ser Gly Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu 130 135 140Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp145 150 155 160Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro 195 200 205Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro 210 215 220Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe225 230 235 240Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 245 250 255Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val 260 265 270Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 275 280 285Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val 290 295 300Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys305 310 315 320Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser 325 330 335Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 340 345 350Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 355 360 365Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375 380Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp385 390 395 400Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp 405 410 415Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 420 425 430Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 435 440 445119446PRTArtificial SequenceHeavy Chain (HC) Sequence 119Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30Glu Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Asp Ile His Pro Gly Ser Gly Gly Thr Ala Asn Asn Gln Lys Phe 50 55 60Lys Gly Arg Val Thr Met Thr Ala Asp Thr Ser Thr Ser Thr Val Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Thr Arg Ala Val Ser Gly Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu 130 135 140Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp145 150 155 160Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro 195 200 205Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro 210 215 220Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe225 230 235 240Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 245 250 255Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val 260 265 270Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 275 280 285Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val 290 295 300Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys305 310 315 320Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser 325 330 335Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 340 345 350Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 355 360 365Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370

375 380Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp385 390 395 400Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp 405 410 415Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 420 425 430Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 435 440 445120446PRTArtificial SequenceHeavy Chain (HC) Sequence 120Glu Val Gln Leu Gln Glu Ser Gly Ala Glu Leu Val Arg Pro Gly Ala1 5 10 15Ser Val Lys Leu Ser Cys Lys Ala Leu Gly Tyr Thr Phe Thr Asp Tyr 20 25 30Glu Ile His Trp Val Lys Glu Thr Pro Val Tyr Gly Leu Glu Trp Ile 35 40 45Gly Asp Ile His Pro Gly Ser Gly Gly Thr Ala Asn Asn Gln Lys Phe 50 55 60Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Asn Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95Thr Arg Ala Val Ser Gly Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110Thr Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu 130 135 140Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp145 150 155 160Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro 195 200 205Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro 210 215 220Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe225 230 235 240Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 245 250 255Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val 260 265 270Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 275 280 285Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val 290 295 300Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys305 310 315 320Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser 325 330 335Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 340 345 350Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 355 360 365Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375 380Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp385 390 395 400Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp 405 410 415Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 420 425 430Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 435 440 445121473PRTArtificial SequenceSIRP alpha sequence 121Glu Glu Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val Ser Val Ala1 5 10 15Ala Gly Glu Ser Ala Ile Leu His Cys Thr Val Thr Ser Leu Ile Pro 20 25 30Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala Arg Glu Leu 35 40 45Ile Tyr Asn Gln Lys Glu Gly His Phe Pro Arg Val Thr Thr Val Ser 50 55 60Glu Ser Thr Lys Arg Glu Asn Met Asp Phe Ser Ile Ser Ile Ser Asn65 70 75 80Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys 85 90 95Gly Ser Pro Asp Thr Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser 100 105 110Val Arg Ala Lys Pro Ser Ala Pro Val Val Ser Gly Pro Ala Ala Arg 115 120 125Ala Thr Pro Gln His Thr Val Ser Phe Thr Cys Glu Ser His Gly Phe 130 135 140Ser Pro Arg Asp Ile Thr Leu Lys Trp Phe Lys Asn Gly Asn Glu Leu145 150 155 160Ser Asp Phe Gln Thr Asn Val Asp Pro Val Gly Glu Ser Val Ser Tyr 165 170 175Ser Ile His Ser Thr Ala Lys Val Val Leu Thr Arg Glu Asp Val His 180 185 190Ser Gln Val Ile Cys Glu Val Ala His Val Thr Leu Gln Gly Asp Pro 195 200 205Leu Arg Gly Thr Ala Asn Leu Ser Glu Thr Ile Arg Val Pro Pro Thr 210 215 220Leu Glu Val Thr Gln Gln Pro Val Arg Ala Glu Asn Gln Val Asn Val225 230 235 240Thr Cys Gln Val Arg Lys Phe Tyr Pro Gln Arg Leu Gln Leu Thr Trp 245 250 255Leu Glu Asn Gly Asn Val Ser Arg Thr Glu Thr Ala Ser Thr Val Thr 260 265 270Glu Asn Lys Asp Gly Thr Tyr Asn Trp Met Ser Trp Leu Leu Val Asn 275 280 285Val Ser Ala His Arg Asp Asp Val Lys Leu Thr Cys Gln Val Glu His 290 295 300Asp Gly Gln Pro Ala Val Ser Lys Ser His Asp Leu Lys Val Ser Ala305 310 315 320His Pro Lys Glu Gln Gly Ser Asn Thr Ala Ala Glu Asn Thr Gly Ser 325 330 335Asn Glu Arg Asn Ile Tyr Ile Val Val Gly Val Val Cys Thr Leu Leu 340 345 350Val Ala Leu Leu Met Ala Ala Leu Tyr Leu Val Arg Ile Arg Gln Lys 355 360 365Lys Ala Gln Gly Ser Thr Ser Ser Thr Arg Leu His Glu Pro Glu Lys 370 375 380Asn Ala Arg Glu Ile Thr Gln Asp Thr Asn Asp Ile Thr Tyr Ala Asp385 390 395 400Leu Asn Leu Pro Lys Gly Lys Lys Pro Ala Pro Gln Ala Ala Glu Pro 405 410 415Asn Asn His Thr Glu Tyr Ala Ser Ile Gln Thr Ser Pro Gln Pro Ala 420 425 430Ser Glu Asp Thr Leu Thr Tyr Ala Asp Leu Asp Met Val His Leu Asn 435 440 445Arg Thr Pro Lys Gln Pro Ala Pro Lys Pro Glu Pro Ser Phe Ser Glu 450 455 460Tyr Ala Ser Val Gln Val Pro Arg Lys465 470122359PRTArtificial SequenceSIRP gamma sequence 122Glu Glu Glu Leu Gln Met Ile Gln Pro Glu Lys Leu Leu Leu Val Thr1 5 10 15Val Gly Lys Thr Ala Thr Leu His Cys Thr Val Thr Ser Leu Leu Pro 20 25 30Val Gly Pro Val Leu Trp Phe Arg Gly Val Gly Pro Gly Arg Glu Leu 35 40 45Ile Tyr Asn Gln Lys Glu Gly His Phe Pro Arg Val Thr Thr Val Ser 50 55 60Asp Leu Thr Lys Arg Asn Asn Met Asp Phe Ser Ile Arg Ile Ser Ser65 70 75 80Ile Thr Pro Ala Asp Val Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys 85 90 95Gly Ser Pro Glu Asn Val Glu Phe Lys Ser Gly Pro Gly Thr Glu Met 100 105 110Ala Leu Gly Ala Lys Pro Ser Ala Pro Val Val Leu Gly Pro Ala Ala 115 120 125Arg Thr Thr Pro Glu His Thr Val Ser Phe Thr Cys Glu Ser His Gly 130 135 140Phe Ser Pro Arg Asp Ile Thr Leu Lys Trp Phe Lys Asn Gly Asn Glu145 150 155 160Leu Ser Asp Phe Gln Thr Asn Val Asp Pro Thr Gly Gln Ser Val Ala 165 170 175Tyr Ser Ile Arg Ser Thr Ala Arg Val Val Leu Asp Pro Trp Asp Val 180 185 190Arg Ser Gln Val Ile Cys Glu Val Ala His Val Thr Leu Gln Gly Asp 195 200 205Pro Leu Arg Gly Thr Ala Asn Leu Ser Glu Ala Ile Arg Val Pro Pro 210 215 220Thr Leu Glu Val Thr Gln Gln Pro Met Arg Val Gly Asn Gln Val Asn225 230 235 240Val Thr Cys Gln Val Arg Lys Phe Tyr Pro Gln Ser Leu Gln Leu Thr 245 250 255Trp Ser Glu Asn Gly Asn Val Cys Gln Arg Glu Thr Ala Ser Thr Leu 260 265 270Thr Glu Asn Lys Asp Gly Thr Tyr Asn Trp Thr Ser Trp Phe Leu Val 275 280 285Asn Ile Ser Asp Gln Arg Asp Asp Val Val Leu Thr Cys Gln Val Lys 290 295 300His Asp Gly Gln Leu Ala Val Ser Lys Arg Leu Ala Leu Glu Val Thr305 310 315 320Val His Gln Lys Asp Gln Ser Ser Asp Ala Thr Pro Gly Pro Ala Ser 325 330 335Ser Leu Thr Ala Leu Leu Leu Ile Ala Val Leu Leu Gly Pro Ile Tyr 340 345 350Val Pro Trp Lys Gln Lys Thr 355123330PRTArtificial SequenceHuman Fc IgG1 Sequence 123Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys1 5 10 15Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr65 70 75 80Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp145 150 155 160Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu225 230 235 240Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr305 310 315 320Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330124330PRTArtificial SequenceHuman Fc IgG1-N297Q Sequence 124Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys1 5 10 15Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr65 70 75 80Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp145 150 155 160Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175Glu Gln Tyr Gln Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu225 230 235 240Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr305 310 315 320Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330125326PRTArtificial SequenceHuman Fc-IgG2 Sequence 125Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg1 5 10 15Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr65 70 75 80Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro 100 105 110Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 115 120 125Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 130 135 140Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly145 150 155 160Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn 165 170 175Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp 180 185 190Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro 195 200 205Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu 210 215 220Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn225 230 235 240Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 245 250 255Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 260 265 270Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 275 280 285Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 290 295 300Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu305 310 315 320Ser Leu Ser Pro Gly Lys 325126377PRTArtificial SequenceHuman Fc-IgG3 Sequence 126Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg1 5 10 15Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr65 70 75 80Tyr Thr Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90

95Arg Val Glu Leu Lys Thr Pro Leu Gly Asp Thr Thr His Thr Cys Pro 100 105 110Arg Cys Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg 115 120 125Cys Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys 130 135 140Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro145 150 155 160Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 165 170 175Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 180 185 190Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Lys Trp Tyr 195 200 205Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 210 215 220Gln Tyr Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Leu His225 230 235 240Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 245 250 255Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln 260 265 270Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met 275 280 285Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 290 295 300Ser Asp Ile Ala Val Glu Trp Glu Ser Ser Gly Gln Pro Glu Asn Asn305 310 315 320Tyr Asn Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu 325 330 335Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Ile 340 345 350Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn Arg Phe Thr Gln 355 360 365Lys Ser Leu Ser Leu Ser Pro Gly Lys 370 375127326PRTArtificial SequenceHuman Fc-IgG4 Sequence 127Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg1 5 10 15Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr65 70 75 80Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro 100 105 110Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 115 120 125Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 130 135 140Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp145 150 155 160Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe 165 170 175Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 180 185 190Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 195 200 205Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 210 215 220Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys225 230 235 240Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 245 250 255Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 260 265 270Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 275 280 285Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 290 295 300Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser305 310 315 320Leu Ser Leu Ser Leu Gly 325128326PRTArtificial SequenceHuman Fc-IgG4 S228P Sequence 128Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg1 5 10 15Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr65 70 75 80Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro 100 105 110Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 115 120 125Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 130 135 140Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp145 150 155 160Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe 165 170 175Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 180 185 190Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 195 200 205Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 210 215 220Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys225 230 235 240Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 245 250 255Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 260 265 270Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 275 280 285Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 290 295 300Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser305 310 315 320Leu Ser Leu Ser Leu Gly 325129327PRTArtificial SequenceHuman Fc-IgG4 PE Sequence 129Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg1 5 10 15Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr65 70 75 80Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro 100 105 110Glu Phe Glu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 115 120 125Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 130 135 140Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp145 150 155 160Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe 165 170 175Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 180 185 190Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 195 200 205Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 210 215 220Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys225 230 235 240Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 245 250 255Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 260 265 270Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 275 280 285Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 290 295 300Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser305 310 315 320Leu Ser Leu Ser Leu Gly Lys 325130326PRTArtificial SequenceHuman Fc-IgG4 PE' Sequence 130Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg1 5 10 15Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr65 70 75 80Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro 100 105 110Glu Phe Glu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 115 120 125Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 130 135 140Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp145 150 155 160Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe 165 170 175Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 180 185 190Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 195 200 205Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 210 215 220Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys225 230 235 240Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 245 250 255Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 260 265 270Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 275 280 285Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 290 295 300Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser305 310 315 320Leu Ser Leu Ser Leu Gly 325131107PRTArtificial SequenceHuman kappa LC Sequence 131Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu1 5 10 15Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu65 70 75 80Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105

Claims

1. A bispecific antibody comprising: a first antigen-binding domain that specifically binds human SIRP.alpha. and comprises a light chain variable domain (VL) and a heavy chain variable domain (VH), and a second antigen-binding domain which specifically binds to a second antigen; wherein the VL comprises LCDR1, LCDR2, LCDR3 sequences selected from: i. SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3; ii. SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6; iii. SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9; iv. SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12; v. SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15; vi. SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18; vii. SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21; viii. SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24; ix. SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27; x. SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30; xi. SEQ ID NO:10, SEQ ID NO:31, SEQ ID NO:12; xii. SEQ ID NO:10, SEQ ID NO:31, SEQ ID NO:32; and xiii. SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27; and wherein the VH comprises HCDR1, HCDR2, HCDR3 sequences selected from: i. SEQ ID NO:33, SEQ ID NO:34, and SEQ ID NO:35; ii. SEQ ID NO:36, SEQ ID NO:37, and SEQ ID NO:38; iii. SEQ ID NO:39, SEQ ID NO:40, and SEQ ID NO:41; iv. SEQ ID NO:42, SEQ ID NO:43, and SEQ ID NO:44; v. SEQ ID NO:45, SEQ ID NO:46, and SEQ ID NO:47; vi. SEQ ID NO:48, SEQ ID NO:49, and SEQ ID NO:50; vii. SEQ ID NO:51, SEQ ID NO:52, and SEQ ID NO:53; viii. SEQ ID NO:54, SEQ ID NO:55, and SEQ ID NO:56; ix. SEQ ID NO:57, SEQ ID NO:58, and SEQ ID NO:59; x. SEQ ID NO:60, SEQ ID NO:61, and SEQ ID NO:62; and xi. SEQ ID NO:57, SEQ ID NO:58, and SEQ ID NO:63.

2. The bispecific antibody of claim 1, wherein the VH comprises the sequence of any one of SEQ ID NOs:81-97 and the VL comprises the sequence of any one of SEQ ID NOs:64-80.

3. The bispecific antibody of claim 1, wherein the wherein the VL comprises the LCDR1, LCDR2, LCDR3 sequences of SEQ ID NOs: 25, 26, and 27, and the VH comprises the HCDR1, HCDR2, and HCDR3 sequences of SEQ ID NOs: 57, 58, and 59.

4. The bispecific antibody of claim 1, wherein the VL comprises the sequence of SEQ ID NO: 72 and the VH comprises the sequence of SEQ ID NO: 89.

5. The bispecific antibody of claim 1, wherein the wherein the VL comprises the LCDR1, LCDR2, LCDR3 sequences of SEQ ID NOs: 10, 11, and 12, and the VH comprises the HCDR1, HCDR2, and HCDR3 sequences of SEQ ID NOs: 42, 43, and 44.

6. The bispecific antibody of claim 1, wherein the VL comprises the sequence of SEQ ID NO: 67 and the VH comprises the sequence of SEQ ID NO: 84.

7. The bispecific antibody of claim 1, wherein the second antigen is selected from CD47, CD19, CD20, CD22, CD24, CD25, CD30, CD33, CD40, CD44, HER2, CD52, CD56, CD70, CD96, CD97, CD99, CD123, CD279 (PD-1), CD117, C-Met, PTHR2, EGFR, RANKL, SLAMF7, PD-L1, CD38, CD19/CD3, HAVCR2 (TIM3), and GD2.

8. The bispecific antibody of claim 1, wherein the antibody is chimeric or humanized.

9. The bispecific antibody of claim 1, wherein the antibody comprises a constant region of the isotype IgG1, IgG1-N297Q, IgG2, IgG4, IgG4-S228P, IgG4-PE or variants thereof.

10. The bispecific antibody of claim 9, wherein the constant region is modified to increase or decrease an antibody effector function selected from antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), antibody-dependent cellular phagocytosis (ADCP), C1q binding, and altered binding to Fc receptors.

11. The bispecific antibody of claim 1, which exhibits anti-tumor activity.

12. The bispecific antibody of claim 1, which increases phagocytosis of human tumor cells.

13. A pharmaceutical composition comprising the bispecific antibody of claim 1, and a pharmaceutically acceptable carrier, diluent, or excipient.

14. A method of treating cancer in a subject in need thereof, the method comprising administering to the subject the bispecific antibody of claim 1, in an amount effective to treat cancer.

15. The method of claim 14, wherein the bispecific antibody is administered in combination with a chemotherapeutic agent.

16. The method of claim 14, wherein the bispecific antibody is administered in combination with an antibody that increases the immune response to cancer.

17. The method of claim 16, wherein the antibody that increases the immune response to cancer inhibits an immune checkpoint or modulates one or more co-stimulatory molecules.

18. The method of claim 14, wherein the bispecific antibody is administered in combination with an opsonizing antibody which targets an antigen on a tumor cell.

19. The method of claim 18, wherein the opsonizing antibody is selected from rituximab (anti-CD20), trastuzumab (anti-HER2), alemtuzumab (anti-CD52), cetuximab (anti-EGFR), panitumumab (anti-EGFR), ofatumumab (anti-CD20), denosumab (anti-RANKL), pertuzumab (anti-HER2), elotuzumab (anti-SLAMF7), atezolizumab (anti-PD-L1), avelumab (anti-PD-L1), durvalumab (anti-PD-L1), necitumumab (anti-EGFR), daratumumab (anti-CD38), obinutuzumab (anti-CD20), blinatumomab (anti-CD19/CD3), and dinutuximab (anti-GD2).

20. The method of claim 14, wherein the cancer is selected from leukemia, lymphoma, multiple myeloma, ovarian cancer, breast cancer, endometrial cancer, colon cancer, rectal cancer, bladder cancer, urothelial cancer, lung cancer, bronchial cancer, bone cancer, prostate cancer, pancreatic cancer, gastric cancer, hepatocellular carcinoma, gall bladder cancer, bile duct cancer, esophageal cancer, renal cell carcinoma, thyroid cancer, head and neck cancer, testicular cancer, cancer of the endocrine gland, cancer of the adrenal gland, cancer of the pituitary gland, cancer of the skin, cancer of soft tissues, cancer of blood vessels, cancer of brain, cancer of nerves, cancer of eyes, cancer of meninges, cancer of oropharynx, cancer of hypopharynx, cancer of cervix, and cancer of uterus, glioblastoma, meduloblastoma, astrocytoma, glioma, meningioma, gastrinoma, neuroblastoma, melanoma, myelodysplastic syndrome, and a sarcoma.

21. The method of claim 14, wherein the cancer is non-small cell lung cancer, adenocarcinoma of the lung, or squamous cell carcinoma of the lung.

22. The method of claim 14, wherein the cancer is selected from systemic mastocytosis, acute lymphocytic (lymphoblastic) leukemia (ALL), T-cell ALL, acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), myeloproliferative disorder/neoplasm, myelodysplastic syndrome, monocytic cell leukemia, and plasma cell leukemia.

23. The method of claim 14, wherein the cancer is a lymphoma selected from histiocytic lymphoma, T-cell lymphoma and B-cell lymphoma.

24. The method of claim 14, wherein the cancer is low-grade/follicular non-Hodgkin's lymphoma (NHL), follicular center cell lymphoma (FCC), mantle cell lymphoma (MCL), diffuse large cell lymphoma (DLCL), small lymphocytic (SL) NHL, intermediate grade/follicular NHL, intermediate grade diffuse NHL, high grade immunoblastic NHL, high grade lymphoblastic NHL, high grade small non-cleaved cell NHL, bulky disease NHL, and Waldenstrom's Macroglobulinemia.

25. The method of claim 14, wherein the cancer is a sarcoma selected from osteosarcoma, Ewing's sarcoma, leiomyosarcoma, synovial sarcoma, alveolar soft part sarcoma, angiosarcoma, liposarcoma, fibrosarcoma, rhabdomyosarcoma, and chrondrosarcoma.