Engineered ph-dependent Anti-CD3 antibodies, and methods for their generation and use

Abstract

To provide: engineered pH-dependent anti-CD3 binding domains and antibodies and / or antigen-binding domains comprising the same, including multispecific antibodies, with, inter alia, desirable T-cell activation potency and (re)directed target cell killing potency and developability profiles; methods for their identification, isolation and generation; and methods for their preparation and use.SOLUTION: The present disclosure relates to engineered pH-dependent anti-CD3 antibodies and antigen-binding fragments thereof, and methods of using the same, where the antibodies and antigen-binding fragments optionally bind to CD3 and / or CD3-expressing cells with greater binding affinities at pH 6.0 than at physiological pH (pH 7.4).SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] Related Applications This application claims priority to U.S. Provisional Patent Application No. 62 / 858,968, filed June 7, 2019, the contents of which are incorporated by reference in their entirety.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy was created on June 3, 2020, is named 1160430.002000.TXT, and is 634,880 bytes in size.

[0003] In particular, the present invention relates to engineered pH-dependent anti-Cluster of Differentiation 3 (CD3) antibodies, including multispecific antibodies, and functional fragments thereof, as well as methods and reagents for their identification, isolation, preparation and use. [Background technology]

[0004] Cell proliferative disorders, such as cancer, are characterized by the uncontrolled proliferation of cell subpopulations. Cell proliferative disorders are the leading cause of death in developed countries and the second leading cause of death in developing countries. The total number of newly diagnosed cases of cancer each year is predicted to reach 23.6 million by 2030. The National Cancer Institute predicts that approximately 2 million new cases of cancer will be diagnosed in the United States in 2018, and that more than 600,000 Americans will die from cancer. Therefore, cancer treatment represents a significant and ever-increasing societal burden.

[0005] The idea of ​​harnessing the cytotoxic ability of T cells to kill tumor cells through the use of CD3-targeting bispecific antibodies dates back to the mid-1980s (Staerz et al. Nature 1985 314:628-32). Many bispecific antibodies developed to date contain a first binding site specific for CD3 for T cell recruitment and activation, and a second binding site for a target disease-associated antigen, such as an antigen produced by tumor cells. By binding to its second target protein expressed on tumors, CD3 bispecific antibodies trigger the CD3 surface receptor on T cells, allowing competent T cells to bind to target-expressing cells via cross-linking by the CD3 bispecific antibody, regardless of the peptide / MHC specificity of their T cell receptors. (See, e.g., Bassan, 2012, Blood 120:5094-95). Using CD3 bispecific antibodies to cross-link T cells and tumor cells can induce dramatic regression of advanced malignancies, even resulting in complete remission in some cases. More than 25 different CD3 bispecific antibodies are currently in clinical development for the treatment of hematological malignancies or solid cancers by targeting CD19, CD20, CD33, and CD123, or EpCAM, HER2, PSMA, and CEA, respectively (see, e.g., Liu et al. Front Immunol 2017 8:38).

[0006] While bispecific antibodies have shown significant advantages over monospecific antibodies for cancer treatment and detection, their widespread commercial application has been hindered by the lack of efficient / low-cost production methods, the lack of stability of bispecific polypeptides, and their short half-life in humans. Many methods for generating bispecific monoclonal antibodies have been developed over the past few decades. However, many bispecific antibody candidates with excellent selectivity and high potency for their target of interest often have challenges in downstream development and clinical efficacy, including: multispecific binding (or "multispecificity"), off-target binding, non-specific binding, poor expression levels or profiles in eukaryotic host cells, e.g., mammalian host cells or yeast cells, poor chemical and physical properties, e.g., poor / low "shelf-life" stability, poor (low) solubility, poor (high) viscosity, tendency to aggregate, and poor clinical and biophysical profiles, e.g., poor pharmacokinetic profile, poor pharmacological profile, fast or poor in vivo clearance rate, short circulatory half-life, some of which cause them to be discontinued in development.

[0007] Specific techniques and assays exist for evaluating many of the aforementioned developability characteristics of discovered antibodies in the context of downstream development activities ("post-development antibodies"), e.g., CIC, SIC, BVP-ELISA, TMA, and other assays. However, such assays are often incompatible with the high-throughput formats of early-stage antibody discovery platforms. Furthermore, these characterizations often require milligram- to gram-scale quantities of protein, which often imposes a practical limit on the number of leads that can be considered for development. This, in turn, reduces the likelihood of program success. As a result, significant resources are often expended attempting to qualify poorly characterized lead candidates, with few backups available in later development stages.

[0008] Various anti-CD3 antibodies, including monoclonal antibodies and bispecific antibodies, are known in the art. For example, see U.S. Patent Nos. 7,262,276, 7,635,472, 7,862,813, 9,587,021, and 10,174,124. However, many of these anti-CD3 antibodies have developmental problems, such as those described above, and / or induce cytokine production, often resulting in harmful cytokine release syndrome (CRS). The anti-CD3 binding domain of bispecific antibodies associates with all T cells, thereby recruiting a subset of highly cytokine-producing CD4 T cells. Thus, there is an unmet need to provide safe and effective anti-CD3 antibodies that exhibit a desirable developability and / or CRS risk profile, e.g., in terms of binding specificity for CD3 expressed on T cells, activation of T cells, and (re)targeting of activated T cells to kill target cells, while attenuating the risk of inducing CRS.

[0009] One approach to developing CD3-binding domains that exhibit a desirable CRS risk profile is to engineer CD3-binding domains with pH-dependent antigen binding. In the past, pH-dependent antigen binding has been engineered by incorporating histidine and / or other ionizable residues into the binding interface of antibodies and other proteins (see, e.g., Igawa et al., Nature Biotechnology 28:1203-1207 (2010)). Protonation of histidine side chains in the binding interface can alter electrostatic interactions and / or induce conformational changes, resulting in pH-dependent differences in binding affinity (Gera et al., PLOS ONE 7(11)e48928.doi:10.1371 / 2012). It is recognized that the pH range of human blood is approximately 7.6–7.8, whereas tumor cells have an extracellular pH of approximately 6.3–6.5. This tumor pH is due, at least in part, to the accumulation of metabolic acids that are not adequately removed due to poor tumor vascularization. Applicant's engineered pH-dependent CD3-binding domain preferentially binds to CD3 at low pH values, promoting binding and activity within and around the tumor microenvironment. Without being bound by theory, it is believed that a CD3-binding domain engineered to preferentially bind to CD3 at low pH, e.g., a pH of about 6, may result in selective and sustained cytotoxic activity at or around the tumor site, potentially reducing or eliminating off-target effects and improving half-life and dosing. Summary of the Invention

[0010] The present disclosure relates to engineered pH-dependent anti-CD3 antibodies, and antigen-binding fragments thereof, and methods of use thereof, which optionally bind to CD3 and / or CD3-expressing cells with higher binding affinity at pH 6.0 than at physiological pH (pH 7.4).

[0011] In certain embodiments, the present disclosure provides an antibody comprising a CD3 binding domain selected from the group consisting of ADI-48576, ADI-48577, ADI-48587, ADI-48592, ADI-48595, ADI-48635, ADI-48643, ADI-48645, ADI-48650, ADI-48652, and ADI-48666.

[0012] In certain embodiments, the present disclosure provides an antibody comprising a CD3 binding domain selected from the group consisting of ADI-48592, ADI-48595, ADI-48650, ADI-48652, ADI-48662, and ADI-48666.

[0013] In certain embodiments, the present disclosure provides an antibody comprising a CD3 binding domain selected from the group consisting of ADI-48588, ADI-48587, ADI-48577, ADI-48590, ADI-48581, ADI-48575, ADI-48593, ADI-48591, ADI-48647, ADI-48636, ADI-48586, ADI-48646, ADI-48638, ADI-48597, ADI-48601, ADI-48576, ADI-48643, ADI-48624, ADI-48632, ADI-48635, and ADI-48645.

[0014] Analysis of 258 unique clones identified using the methods described herein revealed a consensus motif within the CDRH3 region. In some embodiments, the disclosure provides antibodies comprising a CD3-binding domain, CDRH3, wherein the CDRH3-binding domain comprises the consensus motif AX1DX2YX3HX4FYDV, where Xi is R or H, X2 is A or H, X3 is G, H, or P, and X4 is Y, H, D, V, E, S, N, L, M, I, G, A, Q, or T, and optionally, at least one of Xi, X2, X3, and X4 is substituted with H (SEQ ID NO: 1).

[0015] In some embodiments, the disclosure provides antibodies comprising a CD3 binding domain, CDRH3, wherein the CDRH3 binding domain has a consensus motif represented by the sequence ARDX1YGX2X3X4YDX5, where X1 is A or H, X2 is R or H, X3 is H or Y, X4 is F or H, X5 is H or V, and optionally, at least one of X1, X2, X3, X4, and X5 is substituted with H (SEQ ID NO: 2).

[0016] In some embodiments, the disclosure provides an antibody comprising a CD3 binding domain, CDRH3, wherein the CDRH3 binding domain comprises a consensus motif comprising the sequence ARDAHX1X2YX3X4DX5, where X1 is G, E, or R, X2 is R or H, X3 is F or Y, X4 is Y or H, X5 is V or H, and optionally, at least one of X2, X3, X4, and X5 is substituted with H (SEQ ID NO: 3).

[0017] In some embodiments, the disclosure provides an antibody comprising a CD3 binding domain, CDRH3, wherein the CDRH3 binding domain comprises a consensus motif comprising the sequence ARDAX1HRX2FYDV, where X1 is H, Y, S, G, A, T, V, or R, X2 is Y or H, and optionally, at least one of X1 and X2 is substituted with H (SEQ ID NO: 4).

[0018] In some embodiments, the disclosure provides an antibody comprising a CD3-binding domain, CDRH3, wherein the CDRH3-binding domain comprises a consensus motif comprising the sequence ARDX1YHRYFYDX2, where X1 is H or A, X2 is H, V, or M, and optionally, at least one of X1 and X2 is substituted with H (SEQ ID NO: 5).

[0019] In some embodiments, the disclosure provides an antibody comprising a CD3-binding domain, CDRH3, wherein the CDRH3-binding domain comprises a consensus motif comprising the sequence AX1DAYX2X3X4HX5DV, where Xi is R or H, X2 is G or H, X3 is H or R, X4 is N, F or Y, X5 is Y or H, and optionally, at least one of Xi, X2, X3, and X5 is substituted with H (SEQ ID NO: 6).

[0020] In some embodiments, the disclosure provides an antibody comprising a CD3-binding domain, CDRH3, wherein the CDRH3-binding domain comprises a consensus motif comprising the sequence ARDX1X2GRYFYDV, where X1 is M, Q, or H, X2 is R or H, and optionally, at least one of X1 and X2 is substituted with H (SEQ ID NO: 7).

[0021] In some embodiments, the disclosure provides an antibody comprising a CD3 binding domain, CDRH3, wherein the CDRH3 binding domain comprises a consensus motif comprising the sequence ARDX1X2X3RYFYDX4, where X1 is H or A, X2 is T, Y, or H, X3 is G or H, and X4 is V or H, and optionally, at least one of X1, X2, X3, and X4 is substituted with H (SEQ ID NO: 8).

[0022] In some embodiments, the disclosure provides an antibody comprising a CD3-binding domain, CDRH3, wherein the CDRH3-binding domain comprises a consensus motif comprising the sequence AX1DX2X3X4X5X6X7X8DX9, where Xi is R or H, X2 is A, H, M, or Q, X3 is Y, H, S, G, A, T, V, or R, X4 is G, H, P, E, or R, X5 is H or R, X6 is Y, N, F, H, D, E, S, L, M, I, G, A, Q, or T, X7 is F or H, X8 is Y or H, X9 is V, H, or M, and optionally, at least one of Xi, X2, X3, X4, X5, X6, X7, X8, and X9 is H (SEQ ID NO: 58).

[0023] In some embodiments, the disclosure provides an antibody comprising a CD3 binding domain, CDRH3, wherein the CDRH3 binding domain comprises a consensus motif comprising the sequence ARDAX1X2X3X4FYDX5, where Xi is T, H, or Y, X2 is G or H, X3 is H or R, X4 is V or Y, X5 is V or H, and optionally, at least one of Xi, X2, X3, and X5 is substituted with H (SEQ ID NO: 593).

[0024] In some embodiments, the disclosure provides an antibody comprising a CD3 binding domain, CDRH3, wherein the CDRH3 binding domain comprises a consensus motif comprising the sequence AX1DX2X3X4X5X6X7YDX8, where Xi is R or H, X2 is H or A, X3 is H or Y, X4 is H, G or P, X5 is R or H, X6 is Y, I or V, X7 is F or H, and X8 is V or H, and optionally, at least one of Xi, X2, X3, X4, X5, X7 and X8 is substituted with H (SEQ ID NO: 596).

[0025] In some embodiments, the disclosure provides an antibody comprising a CD3-binding domain, CDRH2, wherein the CDRH2-binding domain comprises a consensus motif, the consensus motif comprising the sequence WIDLENANTIYDAKFQG (SEQ ID NO: 9).

[0026] In some embodiments, the disclosure provides antibodies comprising a CD3-binding domain, CDRH2, wherein the CDRH2-binding domain comprises a consensus motif comprising the sequence X1INPX2TGX3TX4YSQKFQG, where X1 is W or Y, X2 is A, S, D, G, N, L, V, H, or Q, X3 is A, T, or S, and X4 is K, V, T, D, Y, F, or A (SEQ ID NO: 10).

[0027] In some embodiments, the disclosure provides an antibody comprising a CD3-binding domain, CDRH2, wherein the CDRH2-binding domain comprises a consensus motif comprising the sequence X1IX2AGTGX3TX4YSQKFQG, where X1 is W, Y, or F, X2 is T, N, or D, X3 is A, T, or L, and X4 is A, K, V, H, T, or N (SEQ ID NO: 11).

[0028] In some embodiments, the disclosure provides an antibody comprising a CD3-binding domain, CDRH2, wherein the CDRH2-binding domain comprises a consensus motif comprising the sequence X1IDAGTGX2TX3YSQKFQG, where X1 is S or W, X2 is L, N, D or F, and X3 is D, Y or K (SEQ ID NO: 12).

[0029] In some embodiments, the disclosure provides an antibody comprising a CD3-binding domain, CDRH2, wherein the CDRH2-binding domain comprises a consensus motif comprising the sequence X1IX2AGTGATX3YSQKFQG, where X1 is G, D, or S, X2 is I or D, and X3 is K or D (SEQ ID NO: 13).

[0030] In some embodiments, the disclosure provides an antibody comprising a CD3-binding domain, wherein the CDRH2-binding domain comprises a consensus motif comprising the sequence WINPX1TGNTX2YSQKFQG, where X1 is D, T, L, S, or A, and X2 is D, V, L, or N (SEQ ID NO: 14).

[0031] In some embodiments, the disclosure provides an antibody comprising a CD3-binding domain, CDRH2, wherein the CDRH2-binding domain comprises a consensus motif comprising the sequence X1INAGTGX2TX3YSQKFQG, where X1 is Y or W, X2 is N, D, or A, and X3 is I or V (SEQ ID NO: 15).

[0032] In some embodiments, the disclosure provides an antibody comprising a CD3-binding domain, CDRH2, wherein the CDRH2-binding domain comprises a consensus motif comprising the sequence X1INPX2TGX3TKYSQKFQG, where X1 is W or Y, X2 is D, I, or Y, and X3 is D, Y, or E (SEQ ID NO: 16).

[0033] In some embodiments, the disclosure provides an antibody comprising a CD3-binding domain, CDRH2, wherein the CDRH2-binding domain comprises a consensus motif comprising the sequence SIX1AGTGX2TKYSQKFQG, where X1 is N or V and X2 is A or I (SEQ ID NO: 17).

[0034] In some embodiments, the disclosure provides an antibody comprising a CD3-binding domain, CDRH2, wherein the CDRH2-binding domain comprises a consensus motif comprising the sequence SINAGTGX1TX2YSQKFQG, where X1 is F or N and X2 is Y or D (SEQ ID NO: 18).

[0035] In some embodiments, the disclosure provides an antibody comprising a CD3-binding domain, CDRH2, wherein the CDRH2-binding domain comprises a consensus motif comprising the sequence X1IX2X3GTGX4TDYSQKFQG, where X1 is D or W, X2 is N or H, X3 is A or S, and X4 is A or N (SEQ ID NO: 19).

[0036] In some embodiments, the disclosure provides an antibody comprising a CD3-binding domain, CDRH2, wherein the CDRH2-binding domain comprises a consensus motif comprising the sequence WIDPX1TGATX2YSQKFQG, where X1 is N, H, or Y, and X2 is V or K (SEQ ID NO: 20).

[0037] In some embodiments, the disclosure provides an antibody comprising a CD3-binding domain, CDRH2, wherein the CDRH2-binding domain comprises a consensus motif comprising the sequence WIX1PX2TGNTKYSQKFQG, where X1 is D or N and X2 is L, I, or V (SEQ ID NO: 21).

[0038] In some embodiments, the disclosure provides an antibody comprising a CD3-binding domain, CDRH2, wherein the CDRH2-binding domain comprises a consensus motif, the consensus motif comprising the sequence SINAGDANTKYSQKFQG (SEQ ID NO: 22).

[0039] In some embodiments, the disclosure provides an antibody comprising a CD3-binding domain, CDRH2, wherein the CDRH2-binding domain comprises a consensus motif comprising the sequence X1IDPX2TGATX3YSQKFQG, where X1 is D or W, X2 is D or V, and X3 is E or D (SEQ ID NO: 23).

[0040] In some embodiments, the disclosure provides an antibody comprising a CD3-binding domain, CDRH2, wherein the CDRH2-binding domain comprises a consensus motif, the consensus motif comprising the sequence WINAGDAATVYSQKFQG (SEQ ID NO: 24).

[0041] In some embodiments, the disclosure provides an antibody comprising a CD3-binding domain, CDRH2, wherein the CDRH2-binding domain comprises a consensus motif comprising the sequence X1IX2X3X4X5X6X7TX8YSQKFQG, where X1 is W, S, Y, F, G, or D, X2 is N, T, D, V, or H, and X3 is A, P, or S. wherein X4 is G, A, S, N, D, L, V, H, Q, T, I or Y, X5 is D or T, X6 is A or G, X7 is A, N, T, S, L, D, F, Y or E, X8 is V, K, T, D, Y, F, A, H, N, L, I or E, and optionally, at least one of Xi, X2, X3, X4, X5, X6, X7 and X8 is H (SEQ ID NO: 59).

[0042] In some embodiments, the disclosure provides an antibody comprising a CD3-binding domain, CDRH2, wherein the CDRH2-binding domain comprises a consensus motif comprising the sequence WIDAGTGX1TX2YSQKFQG, where X1 is L, F, N, or A, and X2 is T or K (SEQ ID NO: 595).

[0043] In some embodiments, the disclosure provides an antibody comprising a CD3-binding domain, CDRH1, wherein the CDRH1-binding domain comprises FNIKDYHMH (SEQ ID NO: 25), SNIKDYYMH (SEQ ID NO: 26), or SNIKDYHMH (SEQ ID NO: 27).

[0044] In some embodiments, the disclosure provides antibodies comprising a CD3-binding domain, CDRH1, wherein the CDRH1-binding domain comprises a consensus motif comprising the sequence YTFX1X2X3X4MH, where Xi is A, K, D, Q, E, N, T, L, Y, S, P, G, H, or V, X2 is T, S, or A, X3 is Y or I, and X4 is A, D, N, S, Y, T, I, V, L, E, P, R, or G (SEQ ID NO: 28).

[0045] In some embodiments, the disclosure provides antibodies comprising a CD3-binding domain, CDRH1, wherein the CDRH1-binding domain comprises a consensus motif comprising the sequence YTFX1X2X3X4MH, where X1 is T, D, A, N, or V, X2 is D, E, G, or Q, X3 is Y or D, and X4 is D, A, E, N, S, Y, or V (SEQ ID NO: 29).

[0046] In some embodiments, the disclosure provides an antibody comprising a CD3-binding domain, CDRH1, wherein the CDRH1-binding domain comprises a consensus motif comprising the sequence YTFTSX1X2MH, where X1 is A, D, or T and X2 is D, F, A, M, V, or Y (SEQ ID NO: 30).

[0047] In some embodiments, the disclosure provides an antibody comprising a CD3-binding domain, CDRH1, wherein the CDRH1-binding domain comprises a consensus motif comprising the sequence YTFX1X2YX3MH, where X1 is N or T, X2 is Q or N, and X3 is S, T, or A (SEQ ID NO: 31).

[0048] In some embodiments, the disclosure provides an antibody comprising a CD3-binding domain, CDRH1, wherein the CDRH1-binding domain comprises a consensus motif comprising the sequence YTFX1X2YVMH, where X1 is I or N and X2 is K or R (SEQ ID NO: 32).

[0049] In some embodiments, the disclosure provides an antibody comprising a CD3-binding domain, CDRH1, wherein the CDRH1-binding domain comprises a consensus motif, the consensus motif comprising the sequence FNIKDYYMH (SEQ ID NO: 47).

[0050] In some embodiments, the disclosure provides an antibody comprising a CD3-binding domain, CDRH1, wherein the CDRH1-binding domain comprises a consensus motif comprising the sequence YTFX1X2YX3MH, where X1 is E, S, or T, X2 is S or D, and X3 is A or D (SEQ ID NO: 31).

[0051] In some embodiments, the disclosure provides an antibody comprising a CD3 binding domain, CDRL3, wherein the CDRL3 binding domain comprises a consensus motif comprising the sequence XI X2SX3X4X5RX6, where XI is H, K, or G, X2 is Q or H, X3 is Y or H, X4 is S, H, D, T, V, M, or L, X5 is R or H, X6 is T or H, and optionally, at least one of XI, X2, X3, X4, X5, and X6 is substituted with H (SEQ ID NO: 33).

[0052] In some embodiments, the disclosure provides an antibody comprising a CD3 binding domain, CDRL3, wherein the CDRL3 binding domain comprises a consensus motif comprising the sequence KQSYX1X2RT, where X1 is H, V, K, W, R, L, G, Y, or Q, and X2 is H, L, E, W, G, M, P, T, Q, or V, and optionally, at least one of X1 and X2 is substituted with H (SEQ ID NO: 34).

[0053] In some embodiments, the disclosure provides an antibody comprising a CD3 binding domain, CDRL3, wherein the CDRL3 binding domain comprises a consensus motif comprising the sequence X1QSX2HX3RT, where X1 is K or H, X2 is H, Y, M, S, L, E, G, or W, X3 is R or K, and optionally, at least one of X1 and X2 is substituted with H (SEQ ID NO: 35).

[0054] In some embodiments, the disclosure provides an antibody comprising a CD3 binding domain, CDRL3, wherein the CDRL3 binding domain comprises a consensus motif comprising the sequence KQSX1X2X3RT, where X1 is Y or H, X2 is T, S, V, or K, and X3 is R or H, and optionally, at least one of X1 and X3 is substituted with H (SEQ ID NO: 36).

[0055] In some embodiments, the disclosure provides an antibody comprising a CD3 binding domain, CDRL3, wherein the CDRL3 binding domain comprises a consensus motif comprising the sequence KQSX1X2X3RT, where X1 is H or Y, X2 is T, S, or Q, and X3 is R or H, and optionally, at least one of X1 and X3 is substituted with H (SEQ ID NO: 36).

[0056] In some embodiments, the disclosure provides an antibody comprising a CD3 binding domain, CDRL3, wherein the CDRL3 binding domain comprises a consensus motif comprising the sequence X1QSX2X3X4RT, where X1 is K or H, X2 is Y or H, X3 is S, H, L, V, or K, and X4 is H, R, or E, and optionally, at least one of X1, X2, X3, and X4 is substituted with H (SEQ ID NO: 598).

[0057] In some embodiments, the disclosure provides an antibody comprising a CD3-binding domain, CDRL2, wherein the CDRL2-binding domain comprises a consensus motif, wherein the consensus motif comprises the sequence WASTRES (SEQ ID NO: 37).

[0058] In some embodiments, the disclosure provides an antibody comprising a CD3-binding domain, CDRL1, wherein the CDRL1-binding domain comprises a consensus motif comprising the sequence KSSQSLLX1X2X3X4GX5NX6LA, where X1 is N or H, X2 is A, R or T, X3 is R or H, X4 is T, P or E, X5 is H or K, X6 is H or Y, and optionally, at least one of X1, X3, X5 and X6 is substituted with H (SEQ ID NO: 38).

[0059] In some embodiments, the present disclosure provides an antibody comprising a CD3 binding domain, CDRL1, wherein the CDRL1 binding domain comprises a consensus motif comprising the sequence KSSQSLLX1AX2THX3NX4LA, where X1 is N or H, X2 is R or H, X3 is K or H, and X4 is Y or H, and optionally, at least one of X1, X2, X3, and X4 is substituted with H (SEQ ID NO: 39).

[0060] In some embodiments, the present disclosure provides an antibody comprising a CD3-binding domain, CDRL1, wherein the CDRL1-binding domain comprises a consensus motif, wherein the consensus motif comprises KSSQSLLNASTAKNYLA (SEQ ID NO: 40) or KSSQSLLNARTRTNYLA (SEQ ID NO: 41).

[0061] In some embodiments, the present disclosure provides an antibody comprising a CD3-binding domain, CDRL1, wherein the CDRL1-binding domain comprises a consensus motif comprising the sequence KSSQSLLNX1X2X3GX4NX5LA, where X1 is S or A, X2 is R or H, X3 is E or T, X4 is H or K, X5 is H or Y, and optionally, at least one of X2, X4, and X5 is substituted with H (SEQ ID NO: 42).

[0062] In some embodiments, the disclosure provides an antibody comprising a CD3-binding domain, CDRL1, wherein the CDRL1-binding domain comprises a consensus motif comprising the sequence KSSQSLLNX1X2TGX3NYLA, where X1 is A or S, X2 is R or H, X3 is H or K, and optionally, at least one of X2 and X3 is substituted with H (sequence number 594).

[0063] In some embodiments, the disclosure provides an antibody comprising a CD3-binding domain, CDRL1, wherein the CDRL1-binding domain comprises a consensus motif comprising the sequence KSSQSLLX1AX2X3X4X5NX6LA, where X1 is N or H, X2 is R or H, X3 is T or E, X4 is G or H, X5 is H or K, X6 is H or Y, and optionally, at least one of X1, X2, X4, X5, and X6 is substituted with H (SEQ ID NO: 597).

[0064] In some embodiments, the disclosure provides a CDRH3 binding domain comprising a consensus motif, the consensus motif comprising the sequence ARDAX1X2X3X4FYDX5, where Xi is T, H, or Y, X2 is G or H, X3 is H or R, X4 is V or Y, X5 is V or H, and optionally, at least one of Xi, X2, X3, and X5 is H (SEQ ID NO: 593); a CDRH2 binding domain comprising a consensus motif, the consensus motif comprising the sequence WIDLENANTIYDAKFQG (SEQ ID NO: 9); a CDRH1 binding domain comprising a consensus motif, the consensus motif comprising the sequence FNIKDYYMH (SEQ ID NO: 47); a CDRL3 binding domain comprising a consensus motif. wherein the consensus motif comprises the sequence KQSX1X2X3RT, where X1 is H or Y, X2 is T, S, or Q, and X3 is R or H, and optionally, at least one of X1 and X3 is H (SEQ ID NO: 36); a CDRH2 binding domain comprising the consensus motif, where the consensus motif comprises the sequence WASTRES (SEQ ID NO: 37); and / or a CDRL1 binding domain comprising the consensus motif, where the consensus motif comprises the sequence KSSQSLLNX1X2TGX3NYLA, where X1 is A or S, X2 is R or H, and X3 is H or K, and optionally, at least one of X2 and X3 is H (SEQ ID NO: 594). In some embodiments, the antibody or antigen-binding fragment is designated as a Group 1 binder, comprising a CD3-binding domain selected from ADI-48592, ADI-48595, ADI-48650, ADI-48652, ADI-48662, and ADI-48666.

[0065] In some embodiments, the disclosure provides a CDRH3 binding domain comprising a consensus motif, the consensus motif comprising the sequence AX1DX2X3X4X5X6X7YDX8, where X1 is R or H, X2 is H or A, X3 is H or Y, X4 is H, G, or P, X5 is R or H, X6 is Y, I, or V, X7 is F or H, and X8 is V or H, and optionally, at least one of X1, X2, X3, X4, X5, X7, and X8 is H. (SEQ ID NO: 596); a CDRH2-binding domain comprising a consensus motif, the consensus motif comprising the sequence WIDLENANTIYDAKFQG (SEQ ID NO: 9) or the sequence WIDAGTGX1TX2YSQKFQG, where X1 is L, F, N, or A, and X2 is T or K (SEQ ID NO: 595); a CDRH1-binding domain comprising a consensus motif, the consensus motif comprising the sequence FNIKDYYMH (SEQ ID NO: 47) or the sequence YTFX1X2YX3MH, where wherein X1 is E, S, or T, X2 is S or D, and X3 is A or D (SEQ ID NO: 31); a CDRL3 binding domain comprising the consensus motif comprising the sequence X1QSX2X3X4RT, wherein X1 is K or H, X2 is Y or H, X3 is S, H, L, V, or K, and X4 is H, R, or E, and optionally, at least one of X1, X2, X3, and X4 is H (SEQ ID NO: 598). a CDRH2-binding domain, wherein the consensus motif comprises the WASTRES sequence (SEQ ID NO: 37); a CDRL1-binding domain comprising a consensus motif, wherein the consensus motif comprises the KSSQSLLX1AX2X3X4X5NX6LA sequence, wherein X1 is N or H, X2 is R or H, X3 is T or E, X4 is G or H, X5 is H or K, and X6 is H or Y; and optionally, at least one of X1, X2, X4, X5, and X6 isH (SEQ ID NO: 597). In some embodiments, the antibody or antigen-binding fragment is designated as a Group 2 binder comprising a CD3-binding domain selected from ADI-48588, ADI-48587, ADI-48577, ADI-48590, ADI-48581, ADI-48575, ADI-48593, ADI-48591, ADI-48647, ADI-48636, ADI-48586, ADI-48646, ADI-48638, ADI-48597, ADI-48601, ADI-48576, ADI-48643, ADI-48624, ADI-48632, ADI-48635, and ADI-48645.

[0066] In some embodiments, the disclosure provides anti-CD3 antibodies or antigen-binding fragments described herein, which, while eliciting T cell activation or T cell killing, exhibit a reduced tendency to elicit cytokine production to levels capable of inducing cytokine release syndrome.

[0067] In some embodiments, the present disclosure provides an anti-CD3 antibody or antigen-binding fragment described herein, wherein the antibody or antigen-binding fragment may comprise a multispecific antibody.

[0068] In some embodiments, the present disclosure provides an anti-CD3 antibody or antigen-binding fragment described herein, wherein the antibody or antigen-binding fragment may comprise a bispecific antibody.

[0069] In some embodiments, the disclosure provides an anti-CD3 antibody or antigen-binding fragment described herein, wherein the antibody or antigen-binding fragment can comprise an scFv.

[0070] In some embodiments, the disclosure provides an anti-CD3 antibody or antigen-binding fragment described herein, wherein the antibody or antigen-binding fragment may comprise at least a second antigen-binding domain that specifically binds to a tumor target, an immune-oncology target, a neurodegenerative disease target, an autoimmune disorder target, an infectious disease target, a metabolic disease target, a cognitive disorder target, a blood-brain barrier target, or a hematological disorder target.

[0071] In some embodiments, the disclosure provides an anti-CD3 antibody or antigen-binding fragment described herein, wherein the antibody or antigen-binding fragment may comprise at least a second antigen-binding domain that specifically binds to an antigen selected from the group consisting of: 17-IA, 4-1BB, 4Dc, 6-keto-PGF1a, 8-iso-PGF2a, 8-oxo-dG, Al adenosine receptor, A33, ACE, ACE-2, activin, activin A, activin AB, activin B, activin C, activin RIA, activin RIA ALK-2, activin RIB. ALK-4, activin RIIA, activin RUB, ADAM, ADAM10, ADAM12, ADAM15, ADAM17 / TACE, ADAM8, ADAM9, ADAMTS, ADAMTS4, ADAMTS5, addressin, aFGF, ALCAM, ALK, ALK-1, ALK-7, alpha-l-antitrypsin, alpha-V / beta-1 antagonist, ANG, Ang, APAF-1, APE, APJ, APP, APRIL, AR, ARC, ART, Artemin, anti-Id, ASPARTIC, atrial natriuretic factor, av / b3 Integrin, Axl, b2M, B7-1, B7-2, B7-H, B-lymphocyte stimulatory factor (BlyS), BACE, BACE-1, Bad, BAFF, BAFF-R, Bag-1, BAK, Bax, BCA-1, BCAM, Bel, BCMA, BDNF, b-ECGF, bFGF, BID, Bik, BFM, BLC, BL-CAM, BLK, BMP, BMP-2 BMP-2a, BMP-3 osteogenin, BMP-4 BMP-2b, BMP-5, BMP-6Vgr-1, BMP-7 (OP-1), BMP-8 (BMP-8a, OP-2), BMPR, BMPR-IA (ALK-3), BMPR-IB (ALK-6), BRK-2, RPK-1, BMPR-II (BRK-3), BMPs, b-NGF, BOK, bombesin, bone-derived neurotrophic factor, BPDE, BPDE-DNA, BTC, complement factor 3 (C3), C3a, C4, C5, C5a, C IO, CA125, CAD-8, calcitonin, cAMP, carcinoembryonic antigen (CEA), tumor-associated antigen, cathepsin A, cathepsin B, cathepsin C / DPPI, cathepsin D, cathepsin E, cathepsin H, cathepsin L, cathepsin O, cathepsin S, cathepsin V, cathepsin X / Z / P, CBL, CCI, CCK2, CCL, CCL1, CCL11, CCL12, CCL13, CCL 14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL2, CCL20, CCL21, CCL22, CCL23, CCL24, CC L25, CCL26, CCL27, CCL28, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9 / 10, CCR, CCR1, C CR10, CCR10, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CD1, CD2, CD4, CD5, CD6, CD7, CD8, CD10, CDlla, CDllb, CDllc, CD13, CD14, CD15, CD16, CD18, CD19, CD20, CD21 , CD22, CD23, CD25, CD27L, CD28, CD29, CD30, CD30L, CD32, CD33 (p67 protein), CD34, CD38, CD40, CD40L, CD44, CD45, CD46, CD49a, CD52, CD54, CD55, CD56, CD61, CD64, CD66e, CD74, CD80 (B7-1), CD89, CD95, CD123, CD137, CD138, CD140a, CD146, CD147, CD148, CD152, CD164, CEACAM5, CFTR, cGMP, CINC, botulinum toxin, Clostridium perfringens toxin, CKb8-l, CLC, CMV, CMVUL, CNTF, CNTN-1, COX, C-Ret, CRG-2, CT-1, CTACK, CTGF, CTLA-4, CX3CL1, CX3CR1, CXCL, CXCL1, CXCL2, CXCL3, CXCL 4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCR, CXCR1, CXCR 2, CXCR3, CXCR4, CXCR5, CXCR6, cytokeratin tumor-associated antigen, DAN, DCC, DcR3, DC-SIGN, decay-accelerating factor, des(l-3)-IGF-I (brain IGF-1), Dhh, digoxin, DNAM-1, Dnase, Dpp, DPPIV / CD26, Dtk, ECAD, EDA, EDA-A1, EDA-A2, EDAR, EGF, EGFR (ErbB-1), EMA, EMMPRIN, EN A, endothelin receptor, enkephalinase, eNOS, Eot, eotaxin, EpCAM, ephrin B2 / EphB4, EPO, ERCC, E-selectin, ET-1, factor Ila, factor VII, factor VIIIc, factor IX, fibroblast activation protein (FAP), Fas, FcRl, FEN-1, ferritin, FGF, FGF-19, FGF-2, FGF3, FGF-8, FGFR, FGFR-3, fibrin, FL, FLIP, Flt-3, Flt-4, follicle-stimulating hormone, fractalkine, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10, G250, Gas 6, GCP-2, GCSF, GD2, GD3, GDF, GDF-1, GDF-3 (Vgr-2), GDF-5 (BMP-14, CDMP-1), GDF-6 (BMP-13, CDMP-2), GDF-7 (BMP-12, CDMP-3), GDF-8 (myostatin), GDF-9, GDF-15 (MIC-1), GDNF, GFAP, GFRa-1, GFR-alpha, GFR-alpha 2, GFR-alpha 3, GITR, glucagon, Glut4, glycoprotein Ilb / IIIa (GP Ilb / IIIa), GM-CSF, gpl30, gp72, GRO, growth hormone-releasing factor, hapten (NP-cap or NIP-cap), HB-EGF, HCC, HCMV gBEnvelope glycoprotein, HCMV gH envelope glycoprotein, HCMV UL, hemopoietic growth factor (HGF), Hep B gpl20, heparanase, Her2, Her2 / neu (ErbB-2), Her3 (ErbB-3), Her4 (ErbB-4), herpes simplex virus (HSV) gB glycoprotein, HSV gD glycoprotein, HGFA, high molecular weight melanoma-associated antigen (HMW-MAA), HIV gpl20, HIV IIIB gp 120 V3 loop, HLA, HLA-DR, HM1.24, HMFGPEM, HRG, Hrk, human cardiac myosin, human cytomegalovirus (HCMV), human growth hormone (HGH), HVEM, 1-309, IAP, ICAM, ICAM-1, ICAM-3, ICE, ICOS, IFNg, Ig, IgA receptor, IgE, IGF, IGF-binding protein, IGF-1R, IGFBP, IGF-I, IGF-II, IL, IL-1, IL-1R, IL -2, IL-2R, IL-4, IL-4R, IL-5, IL-5R, IL-6, IL-6R, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-18, IL-18R, IL-23, interferon (INF)-alpha, INF-beta, INF-gamma, inhibin, iNOS, insulin A-chain, insulin B-chain, insulin-like growth factor 1, Integrin alpha 2, integrin alpha 3, integrin alpha 4, integrin alpha 4 / beta l, integrin, alpha 4 / beta 7, integrin alpha 5 (alpha V), integrin alpha 5 / beta l, integrin alpha 5 / beta 3, integrin alpha 6, integrin beta l, integrin beta 2, interferon gamma, IP-10, l-TAC, JE, kallikrein 2, kallikrein 5, kallikrein 6, kallikrein 11, kallikrein 12, kallikrein 14, kallikrein 15, kallikrein LI, kallikrein L2, kallikrein L3, kallikrein L4, KC, KDR, keratinocyte growth factor (KGF), laminin 5, LAMP, LAP, LAP (TGF-1), latent TGF-1, latent TGF-1bpl, LBP, LDGF, LECT2, Lefty, Lewis-Y antigen, Lewis-Y related antigen, LFA-1, LFA-3, Lfo, LIF, LIGHT, lipoprotein, LIX, LKN, Lptn, L-selectin, LT-a, LT-b, LTB4, LTBP-1, pulmonary surfactant, progesterone, lymphotoxin beta receptor, Mac-1, MAdCAM, MAG, MAP2, MARC, MCAM, MCAM, MCK-2, MCP, M-CSF, MDC, Mer, metalloproteinase, MGDF receptor, MGMT, MHC (HLA-DR), MIF, MIG, MIP, MIP-1-alpha, MK, MMAC1, MMP, MMP-1, MMP-10, MMP-11 MMP-12, MMP-13, MMP-14, MMP-15, MMP-2, MMP-24, MMP-3, MMP-7, MMP-8, MMP-9, MPIF, Mpo, MSK, MSP, mucin (Mucl), MUC18, Müllerian inhibitory factor, Mug, MuSK, NAIP, NAP, NCAD, N-cadherin, NCA90, NCAM, NCAM, neprilysin, neurotrophin-3, -4, or -6, neurturin, nerve growth factor (NGF), NGFR, NGF-beta, nNOS, NO, NOS, Npn, NRG-3, NT, NTN, OB, OGG1, OPG, OPN, OSM, OX40L, OX40R, pl50, p95, PADPr, Parathyroid hormone, PARC, PARP, PBR, PBSF, PCAD, P-cadherin, PCNA, PDGF, PDGF, PDK-1, PECAM, PEM, PF4, PGE, PGF, PGI2, PGJ2, PIN, PLA2, placental alkaline phosphatase (PLAP), PIGF, PLP, PP14, proinsulin, prorelaxin, protein C, PS, PSA, PSCA, prostate-specific membrane antigen (PSMA), PTEN, PTHrp, Ptk, PTN, R51, RANK, RANKL, RANTES, relaxin A-chain, relaxin B-chain, renin, respiratory syncytial virus (RSV) F, RSVFgp, Ret, rheumatoid factor, RLIP76, RPA2, RSK, S100, SCF / KL, SDF-1, SERINE, serum albumin, sFRP-3, Shh, SIGIRR, SK-1, SLAM, SLPI, SMAC, SMDF, SMOH, SOD, SPARC, Stat, STEAP, STEAP-II, TACE, TACI, TAG-72 (tumor-associated glycoprotein-72), TARC, TCA-3, T cell receptor (e.g., T cell receptor alpha / beta), TdT, TECK, TEM1, TEM5, TEM7, TEM8, TERT, testicular PLAP-like alkaline phosphatase, TfR, TGF, TGF-alpha, TGF-beta, TGF-beta pan-specific (Pan Specific), TGF-beta RI (ALK-5), TGF-beta RII, TGF-beta Rllb, TGF-beta RIII, TGF-beta l, TGF-beta 2, TGF-beta 3, TGF-beta 4, TGF-beta 5, thrombin, thymic Ck-1, thyroid-stimulating hormone, Tie, TIMP, TIQ, tissue factor, TMEFF2, Tmpo, TMPRSS2, TNF, TNF-alpha, TNF-alpha beta, TNF-beta 2, TNFc, TNF-RI, TNF-RII, TNFRSF10A (TRAIL Rl Apo-2, DR4), TNFRSFIOB (TRAIL R2 DR5, KILLER, TRICK-2A, TRICK-B), TNFRSF1 0C(TRAIL R3 DcRl、LIT、TRID)、TNFRSF10D(TRAIL R4 DcR2、TRUNDD)、TNFRSF11A(RANK ODF R、TRANCE R)、TNFRSFllB(OPG OCIF、TR1)、TNFRSF12(TWEAK R). FN14)、TNFRSF13B(TACI)、TNFRSF13C(BAFF R)、TNFRSF14(WHEM ATAR、HveA、LIGHT R、TR2)、TNFRSF16(NGFR p75NTR)、TNFRSF17(BCMA)、TNFRSF 18(GITR). AITR)、TNFRSF19(TROY CROWN、TRADE)、TNFRSF19L(RELT)、TNFRSFIA(TNF RI CD120a、p55-60)、TNFRSFIB(TNF RII). CD120b, p75-80, TNFRSF26(TNFRH3), TNFRSF3(LTbR TNF RIII, TNFC R), TNFRSF4(OX40 ACT35, TXGP1 R), TNFRSF 5(CD40 p50), TNFRSF6(Fas). Apo-1, APT1, CD95, TNFRSF6B(DcR3 M68, TR6), TNFRSF7(CD27), TNFRSF8(CD30), TNFRSF9(4-1BB CD137, ILA), TNFRSF21(DR6), TNFRSF22(DcTRAIL R2). TNFRH2, TNFRST23(DcTRAIL Rl TNFRH1), TNFRSF25(DR3 Apo-3, LARD, TR-3, TRAMP, WSL-1), TNFSF10(TRAIL Apo-2 polymer TL2) TNFSF11(TRANCE / RANK dimer ODF) TNFSF12(TWEAK). Apo-3リンド, DR3リンド, TNFSF13(APRIL TALL2), TNFSF13B(BAFF LIGHT, TALL1, THANK, TNFSF20), TNFSF14(LIGHT HVEM diuretic LTg), TNFSF15(TLIA / VEGI), TNFSF18 (GITR diuretic AITR diuretic, TL6), TNFSFIA (TNF-a Connectin, DIF, TNFSF2, TNFSF1B(TNF-b LTa, TNFSF1), TNFSF3(LTb).TNFSF, p33), TNFSF4 (OX40 ligand gp34, TXGP1), TNFSF5 (CD40 ligand CD154, gp39, HIGM1, IMD3, TRAP), TNFSF6 (Fas ligand Apo-1 ligand, APT1 ligand), TNFSF7 (CD27 ligand CD70), TNFSF8 (CD30 ligand CD153), TNFSF9 (4-1BB ligand CD137 ligand), TP-1, t-PA, Tpo, TRAIL, TRAIL R, TRAIL-R1, TRAIL-R2, TRANCE, transfer receptor, TRF, Trk, TROP-2, TSG, TSLP, tumor-associated antigen CA 125, Lewis Y-related glycoprotein-expressing tumor-associated antigen, TWEAK, TXB2, Ung, uPAR, uPAR-1, urokinase, VCAM, VCAM-1, VECAD, VE-cadherin, VE-cadherin-2, VEFGR-1 (flt-1), VEGF, VEGFR, VEGFR-3 (flt-4), VEGI, VFM, viral antigen, VLA, VLA-1, VLA-4, VNR integrin, von V. Revland factor, WIF-1, WNT1, WNT2, WNT2B / 13, WNT3, WNT3A, WNT4, WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9A, WNT9B, WNT10A, WNT10B, WNT11, WNT16, XCL1, XCL2, XCR1, XCR1, XEDAR, XIAP, XPD, CTLA4 (cytotoxic T lymphocyte antigen-4), PD1 (programmed cell death protein 1), PD-L1 (programmed cell death ligand 1), LAG-3 (lymphocyte activation gene-3), TIM-3 (T cell immunoglobulin and mucin protein-3), hormone receptors, and growth factors.

[0072] In some embodiments, the disclosure provides an anti-CD3 antibody or antigen-binding fragment described herein, wherein the antibody or antigen-binding fragment may comprise at least a second antigen-binding domain that specifically binds to an antigen selected from the group consisting of BCMA, cytotoxic T lymphocyte antigen-4 (CTLA4), programmed cell death protein 1 (PD1), programmed cell death ligand 1 (PD-L1), lymphocyte activation agonist (LAG-3), or cytotoxic T lymphocyte antigen-4 (CTLA4). gene-3), TIM-3, CD20, CD2, CD19, Her2, EGFR, EpCAM, FcyRIIIa (CD16), FcyRIIa (CD32a), FcyRIIb (CD32b), FcyRI (CD64), Toll-like receptors (TLRs), TLR4, TLR9, cytokines, IL-2, IL-5, IL-13, IL-6, IL-17, IL-12, IL-23, TNFa, TGFb, cytokine receptors, IL-2R, chemokines, chemokine receptors, growth factors, VEGF, and HGF.

[0073] In some embodiments, the disclosure provides an anti-CD3 antibody or antigen-binding fragment described herein, wherein the antibody or antigen-binding antibody fragment may be contained in a chimeric antigen receptor (CAR), which optionally comprises at least one transmembrane domain and at least one intracellular domain derived from a T cell receptor, and may optionally comprise a CD3 zeta subunit and at least one costimulatory domain.

[0074] In some embodiments, the disclosure provides an anti-CD3 antibody or antigen-binding fragment described herein, wherein the antibody or antigen-binding fragment may comprise scFv2-Fc2 and / or scFv-IgG.

[0075] In some embodiments, the disclosure provides an anti-CD3 antibody or antigen-binding fragment described herein, wherein the antibody or antigen-binding fragment may comprise an IgG constant domain.

[0076] In some embodiments, the disclosure provides an anti-CD3 antibody or antigen-binding fragment described herein, wherein the antibody or antigen-binding antibody fragment optionally comprises at least a second antigen-binding domain that specifically binds to an antigen, wherein the antibody comprises a multispecific format selected from the group consisting of: Fab-Fc-scFv, "bottle opener," Mab-scFv, Mab-Fv, Dual scFv, central Fv, central scFv, one-arm central scFv, Fab-Fab, Fab-Fv, mAb-Fv, mAb-Fab, DART, BiTE, common light chain-IgG, TandAb, Cross-Mab, SEED, BEAT, TrioMab, and DuetMab.

[0077] In some embodiments, the present disclosure provides isolated or recombinant nucleic acid sequences encoding the anti-CD3 antibodies or antigen-binding fragments described herein.

[0078] In some embodiments, the present disclosure provides expression vectors comprising an isolated or recombinant nucleic acid sequence encoding an anti-CD3 antibody or antigen-binding fragment described herein.

[0079] In some embodiments, the present disclosure provides host cells transfected, transformed, or transduced with a nucleic acid sequence encoding an anti-CD3 antibody or antigen-binding fragment described herein, or an expression vector comprising an isolated or recombinant nucleic acid sequence encoding an anti-CD3 antibody or antigen-binding fragment described herein, where the host cell may optionally be a mammalian cell or a yeast cell.

[0080] In some embodiments, the present disclosure provides a pharmaceutical composition comprising an antibody or antigen-binding fragment described herein, or a host cell described herein, and a pharmaceutically acceptable carrier and / or excipient.

[0081] In some embodiments, the present disclosure provides methods of treating a disorder in a mammal in need of such treatment, which may include a proliferative disorder, an oncological disorder, an immune-oncological disorder, a neurological disorder, a neurodegenerative disorder, or an autoimmune disorder, and which may include administering an effective amount of at least one antibody or antigen-binding fragment described herein, or a host cell, optionally an immune cell, and optionally a T cell or an NK cell, expressing one of the antibodies or antigen-binding fragments described herein. In some embodiments, the method may further include administering an additional therapeutic agent to the mammal, optionally in which case the mammal is a human.

[0082] In other embodiments, the present disclosure provides an anti-CD3 antibody or antigen-binding fragment thereof comprising one or more of CDRL1, CDRL2, and CDRL3. Such antibodies, in some embodiments, further comprise CDRH1, CDRH2, and CDRH3. [Brief explanation of the drawings]

[0083] [Figure 1]Figures 1A and 1B show presaturation methods 1 and 2. Figure 1A: CD3 presaturation method #1: Yeast cells were presaturated with native (non-biotinylated) CD3 antigen at pH 7.4 for 10 minutes. The yeast cells were washed at pH 7.4 and incubated in pH 6.0 medium for 10 minutes to dissociate the antigen. Control cells were washed and incubated at pH 7.4. Finally, yeast cells were incubated with biotinylated CD3 antigen (shown as a green circle with a star) at pH 6 for 10 minutes. Control cells were incubated with biotinylated CD3 antigen at pH 7.4. Labeled binders were then sorted and characterized at pH 6. Figure 1B: CD3 presaturation method #2: Yeast cells were presaturated with native CD3 antigen at pH 6.0 for 10 minutes, washed at pH 6.0, and incubated at either pH 7.4 or pH 6.0 for 10 minutes. Finally, yeast cells were incubated with biotinylated CD3 antigen for 10 min at the opposite pH (cells incubated at pH 6.0 in the previous step were incubated at pH 7.4; conversely, cells incubated at pH 7.4 in the previous step were incubated at pH 6.0). Binders labeled with biotinylated CD3 antigen were then sorted and characterized.

[0084] [Figure 2] Figure 2 shows exemplary FACS plots of round 1 and round 2 selections from one library. Similar binding profiles were observed for all libraries. Briefly, during round 1, cells were positively sorted using 100 nM human CD3εδ heterodimer (HuCD3-hd) at pH 6. During round 2, cells were positively sorted using 100 nM HuCD3-hd at pH 6.0 and negatively sorted using 100 nM HuCD3-hd at pH 7.4, or presaturated using method #2 described above. Binding to cynomolgus monkey CD3 (CyCD3-hd) at pH 6.0 was also confirmed. Arrows indicate sorted cells carried over to the next sorting round.

[0085] [Figure 3] Figure 3 shows an exemplary FACS plot from round 3, comparing the input of a pH 6.0 positive sort from round 2 with a pH 7.4 negative sort. Briefly, the sort from round 2 was incubated with 100 nM HuCD3-hd at pH 6.0 and pH 7.4. The overlay column shows that the input cell population (from the round 2 sort) exhibits higher binding at pH 6.0 compared to pH 7.4. Cells were carried forward to the next selection round using the presaturation / toggle method #2.

[0086] [Figure 4] Figure 4 shows exemplary FACS plots from rounds 4 and 5. Round 4 compared cells incubated with 100 nM HuCD3-hd at pH 6 and pH 7.4. Round 4 also compared cells pre-saturated / toggled at pH 6 and pH 7.4. Round 5 compared cells incubated with either 100 nM HuCD3-hd or 100 nM CyCD3-hd at pH 6 (red) and pH 7.4 (gray).

[0087] [Figure 5]Figures 5A and 5B show HuCD3 binding responses. Figure 5A shows the HuCD3 binding responses at pH 6 (x-axis) compared to the HuCD3 binding responses at pH 7.4 (y-axis) for 236 unique clones from the output of round 2 / 3 sorting. Figure 5B shows the KD values ​​of HuCD3 at pH 6 (x-axis) compared to HuCD3 at pH 7.4 (y-axis) for 236 unique clones from the output of round 2 / 3 sorting. Blue circles represent round 2 / 3 clones obtained via presaturation / toggle sorting at pH 6.0, yellow circles represent round 2 / 3 clones obtained via negative sorting at pH 7.4, and red circles represent the parent clone ADI-26906. The results show that negative sorting at pH 7.4 in round 2 / 3 tends to yield more pH-selected binders, but the response or affinity at pH 6.0 was weaker, designated as group 2 binders. Positive selection at pH 6.0 and presaturation / toggle sorting yielded clones with mixed selectivity but high response / affinity. Clones with high affinity at pH 6.0 (e.g., KD < approx. 25 nM) are designated as group 1 binders.

[0088] [Figure 6]Figure 6 shows exemplary kinetics from a ForteBio experiment for four clones compared to the parent clone ADI-26906. The KD for each clone was calculated at pH 7.4 and pH 6.0. The KD ratio was obtained by dividing the KD at pH 7.4 by the KD at pH 6.0. The examples show that some clones designated as Group 1 binders, such as SAD10318_P02_A05 (ADI-48595) and SAD10318_P02_C04 (ADI-48592), bound more strongly (with a lower KD) at pH 6.0 compared to pH 7.4. For example, some clones designated as group 2 binders, such as SAD10318_P01_A03 (ADI-48587) and SAD10318_P01_E01 (ADI-48577), were non-binders at pH 7.4 but bound at pH 6.0. Amino acid substitutions in the CDRH3, CDRL1, and CDRL3 regions that may account for differences in binding are highlighted in the sequence column of Figure 6 (SEQ ID NOS: 576-590, in order of appearance, respectively). For example, clones such as ADI-48576, ADI-48577, ADI-48587, ADI-48592, ADI-48595, ADI-48635, ADI-48650, ADI-48652, ADI-48666, ADI-48643, and ADI-48645 exhibit pH-dependent binding (stronger binding at pH 6.0 compared to binding at pH 7.4), low PSR scores, and offer a broad range of affinity for CD3. DETAILED DESCRIPTION OF THE INVENTION

[0089] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs.As used herein, the term "about" when used in relation to a specific recited numerical value means that the value can vary by 1% or less from the recited value.For example, as used herein, the expression "about 100" includes 99 and 101, and all values ​​therebetween (for example, 99.1, 99.2, 99.3, 99.4, etc.).

[0090] It is to be understood that the aspects and embodiments of the present disclosure described herein include "comprising," "consisting of," and "consisting essentially of" aspects and embodiments.

[0091] Provided herein are anti-CD3 antibodies and antigen-binding fragments thereof that exhibit pH-dependent binding and a favorable developability profile.

[0092] "Cluster of Differentiation 3" or "CD3" generally refers to any native CD3 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), and includes, for example, the CD3ε chain, the CD3γ chain, the CD3α chain, and the CD3β chain, unless otherwise indicated. The term encompasses "full-length," unprocessed CD3 (e.g., unprocessed or unmodified CD3ε or CD3γ) as well as any form of CD3 that results from processing in cells. The term also encompasses naturally occurring variants of CD3, such as splice variants or allelic variants. CD3 includes, for example, the human CD3ε protein (NCBI Reference SEQ ID NO: NP _ 000724), which is a 207 amino acid long protein, and human CD3γ protein (NCBI Reference SEQ ID NO: NP _ 000064), which is 182 amino acids in length. The term also refers to human or cynomolgus monkey CD3ε proteins, SEQ ID NOs: 591 and 592, respectively (Table 4). "CD3εN27" and "CD3εN13" refer to the N-terminal 27 amino acids and the N-terminal 13 amino acids of CD3, respectively, including any chemical modifications or linkages thereto.

[0093] "Anti-CD3 antibody" refers to an antibody or antigen-binding fragment thereof that has the ability to bind to CD3, e.g., CD3ε and / or CD3γ, e.g., human CD3ε and / or CD3γ, with sufficient affinity and / or specificity such that the antibody is useful as a diagnostic and / or therapeutic agent in targeting CD3. In some embodiments, the anti-CD3 antibody has a binding affinity of about 100x10 -9 M or less, about 50x10 -9 M or less, about 25x10 -9 M or less, about 20x10 -9 M or less, or about 10x10 -9 The dissociation constant (K D In some embodiments, the anti-CD3 antibody binds to CD3 at about 5×10 -9 The dissociation constant (K D In some embodiments, the anti-CD3 antibody binds to CD3 at about 2.5 x 10 -9 The dissociation constant (K D In some embodiments, the anti-CD3 antibody binds to CD3 at about 1 x 10 -10 The dissociation constant (K D ) binds to CD3. In some embodiments, K D KD is measured by surface plasmon resonance, e.g., BIACORE, by biolayer interferometry, e.g., using a FORTEBIO Octet HTX instrument (Pall Life Sciences), or by solution-affinity ELISA. In some embodiments, KD is measured using an scFv fragment of an anti-CD3 antibody. In some embodiments, a monovalent KD is measured. In some embodiments, the anti-CD3 antibody binds to an epitope of CD3 that is conserved among CD3 from various species, e.g., the cross-reactive species of human and cynomolgus monkey.

[0094] The term "engineered pH-dependent" refers to an antibody with a modified amino acid sequence that allows for preferential or selective antigen binding at a particular pH. For example, a parent antibody can be engineered (e.g., by modifying the amino acid sequence) to achieve pH-dependent binding. pH-dependent binding refers to the preference of an antibody to bind to an antigen at a given pH (or a given pH range) compared to a different pH (or pH range). In one embodiment, a pH-dependent antibody preferentially or selectively binds to an antigen at a pH of approximately 6 compared to a pH of approximately 7. The antibody sequence may be modified by substitution with one or more ionizable amino acid residues, such as, for example, histidine, lysine, arginine, aspartic acid, and glutamic acid. Ionizable residues may be substituted into the CDRs and / or FRs. In some embodiments, there may be 1 to 10 substitutions per VH or VK of the variant. In some embodiments, there may be 1 to 6 substitutions per VH or VK of the variant.

[0095] The term "cytokine release syndrome" (or CRS) refers to a pro-inflammatory positive feedback loop between cytokines and immune cells, resulting in the excessive and uncontrolled release of pro-inflammatory cytokines by cells within the immune system (see, for example, Lee et al., Blood, Vol. 124, pages 188-195 (2014) and Tisoncik et al., Microbiol Mol Biol Rev, Vol. 76, pages 16-32 (2012)). Upon stimulation and activation, T cells release a range of cytokines at levels and to an extent that produce adverse biological / physiological effects of varying degrees and severity. Such effects include, for example, acute inflammation characterized by redness, swelling or edema, fever, pain, and loss of function. When localized to the skin or other tissues, biological / physiological effects include increased blood flow, allowing vascular leukocytes and plasma proteins to reach extravascular sites of injury, increased local temperature, and the development of pain, tissue edema and extravascular pressure, and decreased tissue perfusion. Other biological / physiological effects include organ and systemic dysfunction, such as heart failure, adult respiratory distress syndrome, neurotoxicity, renal and / or hepatic failure, and disseminated intravascular coagulation. Elevated levels of IFNγ, IL-6, TNFα, TGFβ, IL-2, granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-10, IL-8, IL-5, and / or fractalkine have been implicated as indicators and / or causes of CRS or a propensity for T cell stimulation to induce CRS.

[0096] In certain embodiments, the anti-CD3 antibodies and / or antigen-binding fragments thereof described herein are dumbed down and / or modified to reduce the likelihood or severity of antibody-induced CRS. Non-limiting examples of modifications include silent Fc regions (e.g., removing the Fc entirely or modifying the Fc region to reduce or eliminate effector function) and / or masking (e.g., a polypeptide mask positioned to reduce or inhibit the ability of the antibody or antigen-binding fragment thereof to specifically bind to CD3).

[0097] The term "antibody" is used herein in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and / or antibody fragments (preferably fragments that exhibit the desired antigen-binding activity).

[0098] A "monoclonal antibody" or "mAb" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and / or bind to the same epitope, excluding potential variant antibodies (e.g., containing natural mutations or arising during generation of the monoclonal antibody preparation). Such variants are generally present in small amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen.

[0099] For multispecific antibodies, such antibodies contain at least two different antigen-binding domains that recognize and specifically bind to at least two different antigens. For bispecific antibodies, such antibodies contain two different antigen-binding domains that recognize and specifically bind to at least two different antigens.

[0100] "Different antigens" can refer to different and / or distinct proteins, polypeptides, or molecules, and can also refer to different and / or distinct epitopes, which may be contained within a single protein, polypeptide, or other molecule.

[0101] The term "epitope" refers to an antigenic determinant that interacts with a specific antigen-binding site in the variable region of an antibody molecule, known as the paratope. A single antigen may have multiple epitopes. Thus, different antibodies may bind to different regions of an antigen and have different biological effects. The term "epitope" also refers to the site on an antigen to which B cells and / or T cells respond. It also refers to the region of an antigen that is bound by an antibody. Epitopes may be defined structurally or functionally. Functional epitopes are generally a subset of structural epitopes and contain residues that directly contribute to the affinity of the interaction. Epitopes may also be conformational; that is, composed of amino acids in a nonlinear configuration. In certain embodiments, epitopes may include determinants that are chemically active surface groupings of molecules, such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups. In certain embodiments, epitopes may have specific three-dimensional structural characteristics and / or specific charge characteristics.

[0102] In some instances, antibodies comprise four polypeptide chains: two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, some of which may be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy-chain constant domains that correspond to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0103] In other examples, the antibody may instead comprise a multimeric form thereof (e.g., IgM) or an antigen-binding fragment thereof. Each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region, the heavy chain constant region being composed of domains CH1, CH2, and CH3. Each light chain is composed of a light chain variable region (VL) and a light chain constant region (CL). The VH and VL regions can be further divided into more conserved regions termed framework regions (FR) and regions of hypervariability termed complementarity-determining regions (CDRs) located therebetween. Each VH and VL is composed of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In certain embodiments of the present disclosure, the FRs of the antibody (or antigen-binding fragment thereof) may be identical to human germline sequences, or may be naturally occurring or artificially modified. Consensus amino acid sequences may be defined based on an alignment of two or more CDRs, and thus the heavy chain CDRs are designated "CHRH1," "CDRH2," and "CDRH3," respectively, and the light chain CDRs are designated "CDRL1," "CDRL2," and "CDRL3."

[0104] Unless specifically indicated otherwise, as used herein, the term "antibody" includes molecules comprising two heavy immunoglobulin chains and two light immunoglobulin chains (i.e., "complete antibodies" or "intact antibodies" or "whole antibodies") and antigen-binding fragments thereof.

[0105] An "antigen-binding fragment" refers to a portion of an intact antibody that binds to the antigen to which the intact antibody binds (in this case, CD3). The terms "full length antibody," "intact antibody," and "whole antibody" are used interchangeably herein and refer to an antibody having substantially the same structure as a native antibody.

[0106] Antigen-binding fragments of antibodies include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex, including antibody fragments. Exemplary antigen-binding 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, or only the VH or VL domain), and multispecific antibodies formed from antibody fragments. In some embodiments, the antigen-binding fragment of an anti-CD3 antibody described herein is an scFv.

[0107] As with intact antibody molecules, antigen-binding fragments may be monospecific or multispecific (e.g., bispecific). Multispecific antigen-binding fragments of antibodies may contain at least two different variable domains, in which case each variable domain can specifically bind to a distinct antigen or to different epitopes on the same antigen. In the case of the antigen-binding fragments of anti-CD3 antibodies described herein, a variety of multispecific antibody formats can be used.Non-limiting examples of multispecific and bispecific formats include Fab-Fc-scFv (bottle opener type) (XENCOR), Mab-scFv (XENCOR), Mab-Fv (XENCOR), dual scFv (XENCOR), central Fv (XENCOR), central scFv (XENCOR), one-arm central scFv (XENCOR), Fab-Fab (XENCOR), Fab-Fv (XENCOR), mAb-Fv (XENCOR), mAb-Fab (XENCOR), DART (MACROGENICS), BiTE (AMGEN / MICROMET), KiTE, common light chain-IgG (Genentech), TandAb (SFIMED), Cross-Mab (ROCHE), SEED (EMD Serono), BEAT (Glenmark), TrioMab (Trion Pharma / Fresenius Biotech), DuetMab (Medimune), and the like, which are listed for example in (WO95 / 09917; WO2008 / 119566; WO2008 / 119567; WO2011 / 121110; WO2010 / 037835; WO2007 / 042261; WO2007 / 110205;WO2011 / 121110;WO2012 / 055961;WO2012 / 16067;WO2016 / 086189;WO201 6 / 182751;WO2015 / 006749;WO2014 / 049003;WO2013 / 177101;WO2015 / 128509;US 7,951,917; US 2009 / 0252729; US 2014 / 0348839; US 7,183,076; Mazor et al., Mabs, Vol. 7, pages 377-389 (2015); Muda et al., Protein Engineering, Design, & Selection, Vol. 24, pages 447-454 (2011); and Del Bano et al., Antibodies, Vol. 5, pages 1-23 (2016). In some embodiments, the anti-CD3 scFv fragments described herein comprise one or more variable domains of a multispecific (e.g., bispecific) antibody.

[0108] In certain embodiments, the anti-CD3 antibodies and / or antigen-binding fragments thereof described herein are contained in multispecific antibodies, particularly bispecific antibodies, having binding specificity for a second antigen. Such a second antigen may be an entirely different target from the first target, or may be a different epitope on the same target. In some embodiments, the binding specificities are for two different epitopes of CD3 (e.g., CD3ε or CD3γ). In other embodiments, one of the binding specificities is for CD3 (e.g., CD3ε or CD3γ), and the other is for a different biological molecule (e.g., a cell surface antigen, e.g., a tumor antigen).

[0109] Non-limiting examples of second antigens to which bispecific antibodies, including anti-CD3 antibodies and / or antigen-binding fragments thereof, described herein are directed include targets selected from the group consisting of: 17-IA, 4-1BB, 4Dc, 6-keto-PGF1a, 8-iso-PGF2a, 8-oxo-dG, Al adenosine receptor, A33, ACE, ACE-2, activin, activin A, activin AB, activin B, activin C, activin RIA, activin RIA ALK-2, activin RIB. ALK-4, activin RIIA, activin RUB, ADAM, ADAM10, ADAM12, ADAM15, ADAM17 / TACE, ADAM8, ADAM9, ADAMTS, ADAMTS4, ADAMTS5, addressin, aFGF, ALCAM, ALK, ALK-1, ALK-7, alpha-l-antitrypsin, alpha-V / beta-1 antagonist, ANG, Ang, APAF-1, APE, APJ, APP, APRIL, AR, ARC, ART, Artemin, anti-Id, ASPARTIC, atrial natriuretic factor, av / b3 Integrin, Axl, b2M, B7-1, B7-2, B7-H, B-lymphocyte stimulatory factor (BlyS), BACE, BACE-1, Bad, BAFF, BAFF-R, Bag-1, BAK, Bax, BCA-1, BCAM, Bel, BCMA, BDNF, b-ECGF, bFGF, BID, Bik, BIM, BLC, BL-CAM, BLK, BMP, BMP-2 BMP-2a, BMP-3 osteogenin, BMP-4 BMP-2b, BMP-5, BMP-6Vgr-1, BMP-7 (OP-1), BMP-8 (BMP-8a, OP-2), BMPR, BMPR-IA (ALK-3), BMPR-IB (ALK-6), BRK-2, RPK-1, BMPR-II (BRK-3), BMPs, b-NGF, BOK, bombesin, bone-derived neurotrophic factor, BPDE, BPDE-DNA, BTC, complement factor 3 (C3), C3a, C4, C5, C5a, CIO, CA125, CAD-8, calcitonin, cAMP, carcinoembryonic antigen (CEA), tumor-associated antigen, cathepsin A, catecholamines Cathepsin B, cathepsin C / DPPI, cathepsin D, cathepsin E, cathepsin H, cathepsin L, cathepsin O, cathepsin S, cathepsin V, cathepsin X / Z / P, CBL, CCI, CCK2, CCL, CCLl, CCLll, CCL12, CCL13, CCL14, CCL15, CCL16, CCLl7, CCL18, CCL19, CCL2, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL3, CCL4, CCL5 CCL6, CCL7, CCL8, CCL9 / 10, CCR, CCR1, CCR10, CCR10, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CD1, CD2, CD4, CD5, CD6, CD7, CD8, C D10, CDlla, CDllb, CDllc, CD13, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD27L, CD28, CD29, CD30, CD30L, CD32, CD33( p67 protein), CD34, CD38, CD40, CD40L, CD44, CD45, CD46, CD49a, CD52, CD54, CD55, CD56, CD61, CD64, CD66e, CD74, CD80 (B7-1), CD89, CD95, CD123, CD137, CD138, CD140a, CD146, CD147, CD148, CD152, CD164, CEACAM5, CFTR, cGMP, CINC, botulinum toxin, Clostridium perfringens toxin, CKb8-l, CLC, CMV, CMVUL, CNTF, CNTN-1, COX, C-Ret, CRG-2, CT-1, CTACK, CTGF, CTLA-4, CX3CL1, CX3CR1, CXCL, CXCLl, CXCL2, CXCL3, CXCL 4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCR, CXCR1, CXCR 2, CXCR3, CXCR4, CXCR5, CXCR6, cytokeratin tumor-associated antigen, DAN, DCC, DcR3, DC-SIGN, decay-accelerating factor, des(l-3)-IGF-I (brain IGF-1), Dhh, digoxin, DNAM-1, Dnase, Dpp, DPPIV / CD26, Dtk, ECAD, EDA, EDA-A1, EDA-A2, EDAR, EGF, EGFR (ErbB-1), EMA, EMMPRIN, EN A, endothelin receptor, enkephalinase, eNOS, Eot, eotaxin, EpCAM, ephrin B2 / EphB4, EPO, ERCC, E-selectin, ET-1, factor Ila, factor VII, factor VIIIc, factor IX, fibroblast activation protein (FAP), Fas, FcRl, FEN-1, ferritin, FGF, FGF-19, FGF-2, FGF3, FGF-8, FGFR, FGFR-3, fibrin, FL, FLIP, Flt-3, Flt-4, follicle-stimulating hormone, fractalkine, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10, G250, Gas 6, GCP-2, GCSF, GD2, GD3, GDF, GDF-1, GDF-3 (Vgr-2), GDF-5 (BMP-14, CDMP-1), GDF-6 (BMP-13, CDMP-2), GDF-7 (BMP-12, CDMP-3), GDF-8 (myostatin), GDF-9, GDF-15 (MIC-1), GDNF, GDNF, GFAP, GFRa-1, GFR-alpha, GFR-alpha 2, GFR-alpha 3, GITR, glucagon, Glut 4, glycoprotein Ilb / IIIa (GP Ilb / IIIa), GM-CSF, gpl30, gp72, GRO, growth hormone-releasing factor, hapten (NP-cap or NIP-cap), HB-EGF, HCC, HCMV gBEnvelope glycoprotein, HCMV gH envelope glycoprotein, HCMV UL, hemopoietic growth factor (HGF), Hep B gpl20, heparanase, Her2, Her2 / neu (ErbB-2), Her3 (ErbB-3), Her4 (ErbB-4), herpes simplex virus (HSV) gB glycoprotein, HSV gD glycoprotein, HGFA, high molecular weight melanoma-associated antigen (HMW-MAA), HIV gpl20, HIV IIIB gp 120 V3 loop, HLA, HLA-DR, HM1.24, HMFGPEM, HRG, Hrk, human cardiac myosin, human cytomegalovirus (HCMV), human growth hormone (HGH), HVEM, 1-309, IAP, ICAM, ICAM-1, ICAM-3, ICE, ICOS, IFNg, Ig, IgA receptor, IgE, IGF, IGF-binding protein, IGF-1R, IGFBP, IGF-I, IGF-II, IL, IL-1, IL-1R, IL -2, IL-2R, IL-4, IL-4R, IL-5, IL-5R, IL-6, IL-6R, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-18, IL-18R, IL-23, interferon (INF)-alpha, INF-beta, INF-gamma, inhibin, iNOS, insulin A-chain, insulin B-chain, insulin-like growth factor 1, Integrin alpha 2, integrin alpha 3, integrin alpha 4, integrin alpha 4 / beta l, integrin, alpha 4 / beta 7, integrin alpha 5 (alpha V), integrin alpha 5 / beta l, integrin alpha 5 / beta 3, integrin alpha 6, integrin beta l, integrin beta 2, interferon gamma, IP-10, l-TAC, JE, kallikrein 2, kallikrein 5, kallikrein 6, kallikrein 11, kallikrein 12, kallikrein 14, kallikrein 15, kallikrein LI, kallikrein L2, kallikrein L3, kallikrein L4, KC, KDR, keratinocyte growth factor (KGF), laminin 5, LAMP, LAP, LAP (TGF-1), latent TGF-1, latent TGF-1bpl, LBP, LDGF, LECT2, Lefty, Lewis-Y antigen, Lewis-Y related antigen, LFA-1, LFA-3, Lfo, LIF, LIGHT, lipoprotein, LIX, LKN, Lptn, L-selectin, LT-a, LT-b, LTB4, LTBP-1, pulmonary surfactant, progesterone, lymphotoxin beta receptor, Mac-1, MAdCAM, MAG, MAP2, MARC, MCAM, MCAM, MCK-2, MCP, M-CSF, MDC, Mer, metalloproteinase, MGDF receptor, MGMT, MHC (HLA-DR), MIF, MIG, MIP, MIP-1-alpha, MK, MMAC1, MMP, MMP-1, MMP-10, MMP-11 MMP-12, MMP-13, MMP-14, MMP-15, MMP-2, MMP-24, MMP-3, MMP-7, MMP-8, MMP-9, MPIF, Mpo, MSK, MSP, mucin (Mucl), MUC18, Müllerian inhibitory factor, Mug, MuSK, NAIP, NAP, NCAD, N-cadherin, NCA90, NCAM, NCAM, neprilysin, neurotrophin-3, -4, or -6, neurturin, nerve growth factor (NGF), NGFR, NGF-beta, nNOS, NO, NOS, Npn, NRG-3, NT, NTN, OB, OGG1, OPG, OPN, OSM, OX40L, OX40R, pl50, p95, PADPr, Parathyroid hormone, PARC, PARP, PBR, PBSF, PCAD, P-cadherin, PCNA, PDGF, PDGF, PDK-1, PECAM, PEM, PF4, PGE, PGF, PGI2, PGJ2, PIN, PLA2, placental alkaline phosphatase (PLAP), P1GF, PLP, PP14, proinsulin, prorelaxin, protein C, PS, PSA, PSCA, prostate-specific membrane antigen (PSMA), PTEN, PTHrp, Ptk, PTN, R51, RANK, RANKL, RANTES, relaxin A-chain, relaxin B-chain, renin, respiratory syncytial virus (RSV) F, RSVFgp, Ret, rheumatoid factor, RLIP76, RPA2, RSK, S100, SCF / KL, SDF-1, SERINE, serum albumin, sFRP-3, Shh, SIGIRR, SK-1, SLAM, SLPI, SMAC, SMDF, SMOH, SOD, SPARC, Stat, STEAP, STEAP-II, TACE, TACI, TAG-72 (tumor-associated glycoprotein-72), TARC, TCA-3, T cell receptor (e.g., T cell receptor alpha / beta), TdT, TECK, TEM1, TEM5, TEM7, TEM8, TERT, testicular PLAP-like alkaline phosphatase, TfR, TGF, TGF-alpha, TGF-beta, TGF-beta pan-specific (Pan Specific), TGF-betaRI (ALK-5), TGF-betaRII, TGF-betaRllb, TGF-betaRIII, TGF-beta1, TGF-beta2, TGF-beta3, TGF-beta4, TGF-beta5, thrombin, thymic Ck-1, thyroid-stimulating hormone, Tie, TIMP, TIQ, tissue factor, TMEFF2, Tmpo, TMPRSS2, TNF, TNF-alpha, TNF-alpha beta, TNF-beta2, TNFc, TNF-RI, TNF-RII, TNFRSF10A (TRAIL Rl Apo-2, DR4), TNFRSFIOB (TRAIL R2 DR5, KILLER, TRICK-2A, TRICK-B), TNFRSF10C (TRAIL R3 DcRl, LIT 、TRID)、TNFRSF10D(TRAIL R4 DcR2、TRUNDD)、TNFRSF11A(RANK ODF R、TRANCE R)、TNFRSFllB(OPG OCIF、TR1)、TNFRSF12(TWEAK R). FN14)、TNFRSF13B(TACI)、TNFRSF13C(BAFF R)、TNFRSF14(HVEM ATAR、HveA、LIGHT R、TR2)、TNFRSF16(NGFR p75NTR)、TNFRSF17(BCMA) TNFRSF18(GITR). AITR)、TNFRSF19(TROY CROWN、TRADE)、TNFRSF19L(RELT)、TNFRSFIA(TNF RI CD120a、p55-60)、TNFRSFIB(TNF RII). CD120b, p75-80, TNFRSF26(TNFRH3), TNFRSF3(LTbR TNF RIII, TNFC R), TNFRSF4(OX40 ACT35, TXGP1 R), TNFRSF5(CD40 p50), TNFRSF6(Fas). Apo-1, APT1, CD95, TNFRSF6B(DcR3 M68, TR6), TNFRSF7(CD27), TNFRSF8(CD30), TNFRSF9(4-lBB CD137, ILA), TNFRSF21(DR6), TNFRSF22(DcTRAIL R2). TNFRH2, TNFRST23(DcTRAIL Rl TNFRH1), TNFRSF25(DR3 Apo-3, LARD, TR-3, TRAMP, WSL-1), TNFSF10(TRAIL Apo-2 polymer TL2) TNFSF11(TRANCE / RANK dimer ODF) TNFSF12(TWEAK). Apo-3リンド, DR3リンド, TNFSF13(APRIL TALL2), TNFSF13B(BAFF LIGHT, TALL1, THANK, TNFSF20), TNFSF14(LIGHT HVEM ligand LTg) TNFSF15(TL1A / VEGI) TNFSF18(GITR ligand AITR ligand TL6) TNFSFIA(TNF-a Connectin, DIF, TNFSF2, TNFSF1B (TNF-b LTa, TNFSF1), TNFSF3 (LTb TNFC, p33), TNFSF4 (OX40 dimer).gp34, TXGP1), TNFSF5 (CD40 ligand CD154, gp39, HIGM1, IMD3, TRAP), TNFSF6 (Fas ligand Apo-1 ligand, APT1 ligand), TNFSF7 (CD27 ligand CD70), TNFSF8 (CD30 ligand CD153), TNFSF9 (4-lBB ligand CD137 ligand), TP-1, t-PA, Tpo, TRAIL, TRAIL R, TRAIL-R1, TRAIL-R2, TRANCE, transfer receptor, TRF, Trk, TROP-2, TSG, TSLP, tumor-associated antigen CA 125, Lewis Y-related glycoprotein-expressing tumor-associated antigen, TWEAK, TXB2, Ung, uPAR, uPAR-1, urokinase, VCAM, VCAM-1, VECAD, VE-cadherin, VE-cadherin-2, VEFGR-1 (flt-1), VEGF, VEGFR, VEGFR-3 (flt-4), VEGI, VIM, viral antigen, VLA, VLA-1, VLA-4, VNR integrin, von V. Revland factor, WIF-1, WNT1, WNT2, WNT2B / 13, WNT3, WNT3A, WNT4, WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9A, WNT9B, WNT10A, WNT10B, WNT11, WNT16, XCL1, XCL2, XCR1, XCR1, XEDAR, XIAP, XPD, CTLA4 (cytotoxic T lymphocyte antigen-4), PD1 (programmed cell death protein 1), PD-L1 (programmed cell death ligand 1), LAG-3 (lymphocyte activation gene-3), TIM-3 (T cell immunoglobulin and mucin protein-3), hormone receptors, and growth factors.

[0110] Multispecific agents, including anti-CD3 antibodies and antigen-binding fragments disclosed herein, can be engineered using a variety of techniques, including, but not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chains with different specificities (see, e.g., Milstein and Cuello, Nature 305:537 (1983)), WO 93 / 08829, and Traunecker et al., EMBO J. 10:3655 (1991)), "knobs-in-holes" engineering (see, e.g., U.S. Pat. No. 5,731,168), immunoglobulin crossover (also known as Fab domain swapping or CrossMab format) technology (see, e.g., WO 2009 / 080253; Schaefer et al., EMBO J. 10:3655 (1991)), and other techniques. al., Proc. Natl. Acad. Sci. USA, 108:11187-11192 (2011)), engineering electrostatic steering effects for the creation of antibody Fc heterodimeric molecules (WO2009 / 089004A1), cross-linking of two or more antibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980, and Brennan et al., Science, 229:81 (1985)), leucine zippers (see, e.g., Kostelny et al., J. Immunol, 148(5):1547-1553 (1992)), "diabody" technology (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)), single-chain Fv (sFv) dimers (see, e.g., Gruber et al., J. Immunol, 152:5368 (1994)), and trispecific antibodies, for example, as described in Tutt et al. J. Immunol 147:60 (1991).

[0111] The present disclosure also contemplates modifications of the anti-CD3 antibodies disclosed herein, including substitutions, insertions, and / or deletions of one or more amino acids in the FR and / or CDR regions of the heavy and light chain variable domains. Once obtained, such derived antibodies and / or antigen-binding fragments may be tested for one or more desirable properties, such as improved binding specificity, increased binding affinity, improved developability, etc.

[0112] In some embodiments, the anti-CD3 antibody and / or antigen-binding fragment thereof comprises a heavy chain (HC) sequence, a light chain (LC) sequence, a CDRH3 sequence, a CDRH2 sequence, a CDRH1 sequence, a CDRL3 sequence, a CDRL2 sequence, a CDRL1 sequence, and / or a framework sequence. In some embodiments, the anti-CD3 antibodies and / or antigen-binding fragments thereof have amino acid sequence identity of at least about 100%, at least about 99%, at least about 98%, at least about 97%, at least about 96%, at least about 95%, at least about 94%, at least about 93%, at least about 92%, at least about 91%, at least about 90%, at least about 89%, at least about 88%, at least about 87%, at least about 86%, at least about 85%, at least about 84%, at least about 83%, at least about 82%, at least about 80%, and / or any percentage of identity therebetween, to the corresponding sequences of the anti-CD3 antibodies disclosed in Table 1 (Ab1-Ab258). In some embodiments, percent identity is measured by any known algorithm for sequence identity, such as, for example, FASTA, BLAST, or GAP.

[0113] In some embodiments, residue positions that are not identical differ by conservative amino acid substitutions. A "conservative amino acid substitution" is one in which an amino acid residue is replaced with another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). Generally, conservative amino acid substitutions do not substantially change the functional properties of a protein. When two or more amino acid sequences differ from each other by conservative substitutions, the percent or degree of similarity may be adjusted upward to correct for the conservative nature of the substitution. Means for making this adjustment are known to those of skill in the art. (See, e.g., Pearson (1994) Methods Mol. Biol. 24:307-331.) Examples of groups of amino acids with side chains having similar chemical properties include: 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine, 2) aliphatic-hydroxyl side chains: serine and threonine, 3) amide-containing side chains: asparagine and glutamine, 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan, 5) basic side chains: lysine, arginine, and histidine, 6) acidic side chains: aspartic acid and glutamic acid, and 7) sulfur-containing side chains: cysteine ​​and methionine. In some embodiments, conservative amino acid substitution groups are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine. Alternatively, in some embodiments, a conservative substitution includes any change that has a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256:1443 45. In some embodiments, a "moderately conservative" substitution includes any change that has a non-negative value in the PAM250 log-likelihood matrix.

[0114] Substitution of one or more CDR residues or omission of one or more CDRs is also possible. The scientific literature reports antibodies in which one or two CDRs can be deleted to alter binding. Padlan et al. (1995 FASEB J. 9:133-139) analyzed the contact regions between antibodies and their antigens based on published crystal structures and concluded that only approximately one-fifth to one-third of the CDR residues actually contact their associated antigen. Padlan also found that in many antibodies, one or two CDRs do not contain any amino acids that contact the antigen (see also Vajdos et al. 2002 J Mol Biol 320:415-428). CDR residues that do not contact the antigen can be identified by molecular modeling and / or empirically based on previous studies from regions of the Kabat CDRs outside the Chothia CDRs (e.g., residues H60-H65 of CDRH2 are often not required). When a CDR or residue thereof is omitted, it is usually replaced with an amino acid at the corresponding position in another human antibody sequence or a consensus of such sequences. The position of substitution within the CDR, and the amino acid to be substituted, can also be selected empirically.

[0115] In certain embodiments, substitutions, insertions, or deletions may be present in one or more CDRs of the engineered pH-dependent CD3-binding antibodies described herein, so long as such changes maintain pH sensitivity and do not substantially reduce the ability of the antibody to bind to its antigen. For example, conservative changes (e.g., conservative substitutions provided herein) that do not substantially reduce binding affinity may be made in a CDR. Such changes may, for example, be outside of the antigen-contacting residues in the CDR. In certain embodiments of the variant VH and VL sequences provided above, each CDR contains no changes or no more than one, two, or three amino acid substitutions.

[0116] A useful method for identifying antibody residues or regions that can be targeted for mutagenesis is called "alanine scanning mutagenesis," described in Cunningham and Wells (1989) Science, 244:1081-1085. In this method, a residue or group of target residues (e.g., charged residues such as arg, asp, his, lys, and glu) is identified and replaced with neutral or negatively charged amino acids (e.g., alanine or polyalanine), and it is determined whether the antibody-antigen interaction is affected. Further substitutions can be introduced at amino acid positions that show functional sensitivity to the initial substitution. Alternatively, or in addition, crystal structures of antigen-antibody complexes can be used to identify contact points between the antibody and antigen. Such contact residues and adjacent residues can be targeted or excluded from substitution candidates. Variants can be screened to determine whether they contain desirable properties.

[0117] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing several hundred or more residues, as well as intersequence insertions of single or multiple amino acid residues. An example of a terminal insertion is an antibody with an N-terminal methionyl residue. Other insertional variants of antibody molecules include the fusion to the antibody N- or C-terminus to an enzyme (e.g., an enzyme for ADEPT) or a polypeptide that increases the serum half-life of the antibody.

[0118] As described throughout, the anti-CD3 antibodies and / or antigen-binding fragments thereof provided herein have favorable developability and are therefore relatively amenable to development.

[0119] The term "developable" refers to the extent to which one or more polypeptides in a plurality of polypeptides possess desirable properties, such as, for example, desirable expression in mammalian cells, solubility, viscosity, aggregation, chemical and / or physical stability, a desirable shelf life, melting point, pharmacokinetic profile, circulating half-life, and clearance characteristics. Such properties may be treated individually, as a combination of a subset of such properties, or collectively, as indicators of the likelihood that the one or more polypeptides will be successfully developed as therapeutic candidates and ultimately become approved drugs. Thus, as understood in the art, polypeptides having desirable developability properties generally possess, for example, relatively high solubility, relatively low viscosity, relatively low tendency to aggregate, relatively high chemical stability, relatively high physical stability, a relatively long shelf life, a relatively high melting point, a relatively long circulating half-life, a relatively long clearance time, etc. Polypeptides with undesirable developability properties possess, for example, relatively low solubility, relatively high viscosity, relatively high tendency to aggregate, relatively poor chemical stability, relatively poor physical stability, relatively short shelf life, relatively low melting point, relatively short circulatory half-life, relatively long clearance time, etc.

[0120] Methods and assays that can be employed to confirm the extent to which a polypeptide, such as, for example, an anti-CD3 antibody and / or antigen-binding fragment thereof described herein, possesses desirable developability properties are available in the art, such as, for example, PSR assays (WO2014 / 179363, and Xu et al., Protein Eng Des Sel, Vol. 26, pages 663-670 (2013)), SMP assays and SCP assays, cross-interaction chromatography (CIC), self-interaction chromatography (SIC), dynamic light scattering, size-exclusion chromatography (SEC), dynamic light scattering (DLS) spectroscopy, photon correlation spectroscopy, quasi-elastic light scattering, circular dichroism (CD), viscosity measurements, whole cell binding, tissue microarray methodology, BVP ELISA assays, AC-SINS assays (Liu et al; MAbs, Vol. 6, 483-492 (2014)); differential scanning calorimetry, and the like (e.g., He et al. al.,J.Pharm.Sci.,Vol.100(4),pp.1330-1340(2011);Wagner et al.,Pharm.Develop.& Technol(Posted online in 2012;hyper-text transfer protocol:informahealthcare.com / doi / abs / 10.3109 / 10837450.2011.649851);Hotzel et al.,MAbs,Vol.4(6),753-7601(2012);Weiqiang et al. al.,J.Pharm.Sci.,Vol.101(5),pp.1701-1720(2012);Banks et al.,J.Pharm.Sci.,Vol.101(8),pp.2720-2732(2012);Lie et al. al., J. Pharm. Sci., Vol. 94(9), pp. 1928-1948 (2005); and Payne et al., Biopolymers, Vol. 85(5), pp. 527-533 (2006).

[0121] In some embodiments, antibodies identified as having low developability are so detected by their interaction with a multispecific reagent (PSR) and are therefore referred to as "multispecific" polypeptides. Such multispecific antibodies may also be referred to as relatively "undevelopable" or relatively "non-developable."

[0122] A "developability profile" refers to an index that can be assigned to an antibody when assessing its developability. A developability profile is a scale or metric by which the developability of anti-CD3 antibodies can be evaluated, compared, and / or ranked. Such a developability profile serves as a measure of the degree of interaction between a CD3-binding substance and an antibody comprising the same. The degree of interaction may be assessed by any number of means available in the art that provide an output value that correlates with the strength or affinity of a polypeptide for a conjugated moiety. Exemplary means include, for example, flow cytometry such as FACS, ELISA, quantitative immunoaffinity assays, immunoprecipitation assays, mammalian two-hybrid assays, or yeast two-hybrid assays. In the case of FACS, as shown in the examples, the degree of interaction between a polypeptide in a plurality of polypeptides and PSR may be ascertained by generating a mean fluorescence intensity (MFI) for each detected polypeptide-PSR interaction, then ordering the MFIs in either ascending or descending order, thereby ranking the polypeptides in the plurality of polypeptides according to the relative degree of interaction between each detected polypeptide and PSR. By performing such a ranking on a plurality of polypeptides, polypeptides with high developability can be easily identified, and polypeptides with low developability can also be easily identified.

[0123] The developability profile may also take the form of a normalized score, for example, by normalizing the developability of an anti-CD3 antibody described herein to the developability of a standard (or control) antibody, such as an anti-HEL antibody.

[0124] In certain embodiments, the engineered pH-dependent CD3-binding domains of the present invention and antibodies comprising the same may be further modified to contain additional nonproteinaceous moieties known and readily available in the art. Suitable moieties for derivatization of antibodies include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone), polyethylene glycol, polypropylene glycol homopolymer, polypropylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and combinations thereof. Polyethylene glycol propionaldehyde may be advantageous in manufacturing due to its stability in water. The polymer may be of any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody may vary, and when multiple polymers are attached, they may be the same molecule or different molecules. Generally, the number and / or type of polymers used for derivatization can be determined depending on considerations including, but not limited to, the particular property or function of the antibody to be improved, whether the antibody derivative will be used therapeutically under defined conditions, etc.

[0125] In certain embodiments, the engineered pH-dependent CD3 binding domains and antibodies comprising the same exhibit enhanced developability profiles. The developability profile of an anti-CD3 antibody is obtained by performing one or more of a PSR assay, a SCP assay, an AC-SINS assay, an ELISA, a DSF assay, a Tm assay, a HIC assay, a CIC assay, or a combination thereof.

[0126] In other embodiments, the anti-CD3 antibodies and / or antigen-binding fragments thereof described herein exhibit a poly-specificity reagent (PSR) score of about 0.0 to about 0.45. In some embodiments, the PSR is about 0.0 to about 0.4. In some embodiments, the PSR is about 0.0 to about 0.35. In some embodiments, the PSR is about 0.0 to about 0.3. In some embodiments, the PSR is about 0.0 to about 0.25. In some embodiments, the PSR is about 0.0 to about 0.2. In some embodiments, the PSR is about 0.0 to about 0.15. In some embodiments, the PSR is about 0.0 to about 0.1. In some embodiments, a score of 0.0 to 0.1 is a "clean PSR." In some embodiments, a score of 0.1 to 0.33 is a "low PSR." In some embodiments, a score of 0.33 to 0.66 is a "medium PSR." In some embodiments, a score of 0.66 to 1.00 is a "high PSR." In some embodiments, a high PSR score indicates reduced (or poor) developability. Generally, the lower the PSR score, the more favorable the developability of the antibody.

[0127] In still other embodiments, the anti-CD3 antibodies and / or antigen-binding fragments thereof described herein exhibit an HIC score of less than about 10.5 minutes (clean to low HIC score). In some embodiments, the HIC score is between about 10.5 minutes and 11.5 minutes (medium HIC score). In some embodiments, the HIC score is greater than about 11.5 minutes (high HIC score). Generally, the lower the HIC score, the more favorable the antibody's developability.

[0128] In still other embodiments, the anti-CD3 antibodies and / or antigen-binding fragments thereof described herein exhibit an SEC score of less than about 95%, indicating that the antibody is monomeric, i.e., not aggregated.

[0129] In still other embodiments, the anti-CD3 antibodies and / or antigen-binding fragments thereof described herein exhibit a Tm of less than about 65°C.

[0130] In some embodiments, the anti-CD3 antibodies and / or antigen-binding fragments thereof described herein may be further modified to minimize effector function, e.g., a silenced Fc.

[0131] "Effector function" refers to a biological activity attributable to the Fc region of an antibody and varies depending on the antibody isotype. Exemplary effector functions include C1q binding, complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, down-regulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

[0132] The "Fc region" is the C-terminal region of an immunoglobulin heavy chain containing at least a portion of the constant region, including native-sequence Fc regions and variant Fc regions. The Fc region of a human IgG heavy chain can range from Cys226 or from Pro230 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may be present or absent. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region follows the EU numbering system, also known as the EU index. This system is described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991.

[0133] In certain embodiments, one or more amino acid modifications may be introduced into the Fc region of an anti-CD3 antibody of the disclosure, thereby generating an Fc region variant (see, e.g., US2012 / 0251531). The Fc region variant may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) containing an amino acid modification (e.g., substitution) at one or more amino acid positions.

[0134] In certain embodiments, the present disclosure contemplates anti-CD3 antibody variants that retain some, but not all, effector functions, making them desirable candidates for applications in which in vivo antibody half-life is important but certain effector functions (e.g., complement and ADCC) are unnecessary or deleterious. In vitro and / or in vivo cytotoxicity assays can be performed to confirm the reduction / loss of CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to confirm that the antibody lacks FcγR binding (and thus likely lacks ADCC activity) but retains FcRn binding ability. Primary cells that mediate ADCC (e.g., NK cells) express only FcγIII, whereas monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays for assessing ADCC activity of a molecule of interest are described in U.S. Pat. No. 5,500,362 (e.g., Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); U.S. Pat. No. 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assay methods may be employed (see, e.g., ACTI™ Non-Radioactive Cytotoxicity Assay for Flow Cytometry (Cell Technology, Inc., Mountain View, CA); and CytoTox 96® Non-Radioactive Cytotoxicity Assay (Promega, Madison, WI)). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer cells (NK).Alternatively, or in addition, the ADCC activity of the molecule of interest may be assessed in vivo in an animal model, such as that disclosed in Clynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). A C1q binding assay may be performed to confirm that the antibody is unable to bind to C1q and therefore lacks CDC activity. See, for example, the C1q and C3c binding ELISAs in WO2006 / 029879 and WO2005 / 100402. To assess complement activation, a CDC assay may be performed (see, e.g., Gazzano-Santoro et al. J. Immunol Methods 202:163 (1996); Cragg, MS et al. Blood. 101:1045-1052 (2003); and Cragg, MS and MJ Glennie Blood. 103:2738-2743 (2004)). FcRn binding and in vivo clearance / half-life determinations can also be performed using methods known in the art (see, e.g., Petkova, SB et al. Int'l. Immunol 18(12):1759-1769 (2006)).

[0135] In some embodiments, antibodies with reduced effector function include antibodies with substitutions at one or more of residues 238, 265, 269, 270, 297, 327, and 329 in the Fc region (U.S. Patent Nos. 6,737,056 and 8,219,149). In some embodiments, Fc variants include Fc variants with substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc variant in which residues 265 and 297 are substituted with alanine (U.S. Patent Nos. 7,332,581 and 8,219,149).

[0136] In other embodiments, the anti-CD3 antibodies and / or antigen-binding fragments thereof described herein are further modified to include a masking agent, such as a polypeptide mask attached via a cleavable linker.

[0137] In some embodiments, the anti-CD3 antibodies and / or antigen-binding fragments thereof described herein are conjugated to a therapeutic moiety, thereby forming an immunoconjugate. An "immunoconjugate" is an antibody conjugated to one or more heterologous molecules, such as, for example, an antibiotic, a second anti-CD3 antibody, a vaccine, or a toxoid, or any other therapeutic moiety.

[0138] In certain embodiments, the anti-CD3 antibodies and / or antigen-binding fragments thereof described herein are modified to increase or decrease the extent to which the antibodies are glycosylated. Adding or deleting glycosylation sites from an anti-CD3 antibody of the present disclosure can be readily accomplished by altering the amino acid sequence to create or remove one or more glycosylation sites.

[0139] Preparation of anti-CD3 antibodies and their antigen-binding fragments Anti-CD3 antibodies and / or antigen-binding fragments thereof may be produced using recombinant methods. For example, isolated nucleic acids encoding the anti-CD3 antibodies described herein are provided. The nucleic acids may encode an amino acid sequence comprising the antibody VL and / or an amino acid sequence comprising the antibody VH (e.g., the antibody light chain and / or heavy chain). In further embodiments, one or more vectors (e.g., expression vectors) comprising such nucleic acids are provided. In further embodiments, host cells comprising such nucleic acids are provided. In one such embodiment, the host cell comprises (e.g., is transformed with) (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising the antibody VL and an amino acid sequence comprising the antibody VH, or (2) a first vector comprising a nucleic acid encoding an amino acid sequence comprising the antibody VL and a second vector comprising a nucleic acid encoding an amino acid sequence comprising the antibody VH. In one embodiment, the host cell is a eukaryotic cell, such as a Chinese hamster ovary (CHO) cell or a lymphoid cell (e.g., a Y0, NS0, or Sp20 cell). In one embodiment, a method of producing an anti-CD3 antibody is provided, the method comprising culturing a host cell containing nucleic acid encoding the antibody, as described above, under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium).

[0140] The term "host cell" refers to a cell into which exogenous nucleic acid has been introduced, including the progeny of such a cell. Host cells include transformants and transformed cells, which include the primary transformed cell and its progeny without regard to the number of transfers.

[0141] For recombinant production of an anti-CD3 antibody, nucleic acid encoding the antibody is isolated, e.g., as described above, and inserted into one or more vectors for further cloning and / or expression in host cells. The nucleic acid can be readily isolated and sequenced using conventional methods (e.g., by using oligonucleotide probes capable of binding specifically to genes encoding the antibody heavy and light chains).

[0142] Suitable host cells for cloning or expressing antibody-encoding vectors include prokaryotic or eukaryotic cells. For example, antibodies can be produced in bacteria, particularly if glycosylation and Fc effector functions are not required. For the expression of antibody fragments and polypeptides in bacteria, see, e.g., U.S. Patent Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ, 2003), pp. 245-254, which describes the expression of antibody fragments in E. coli.) After expression, the antibody can be isolated from the bacterial cell paste in a soluble fraction and can also be further purified. In addition to prokaryotes, eukaryotes such as filamentous fungi or yeast, including fungal and yeast strains whose glycosylation pathways have been "humanized" to produce antibodies with partially or fully human glycosylation patterns, are suitable cloning or expression hosts for antibody-encoding vectors. See, e.g., Gerngross, Nat. Biotech. 22:1409-1414 (2004), and Li et al., Nat. Biotech. 24:210-215 (2006); WO2009 / 036379; WO2010 / 105256; and WO2012 / 009568.

[0143] Plant cell cultures can also be used as hosts.See, for example, U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (which describe the PLANTIBODY™ technology for producing antibodies in transgenic plants).Vertebrate cells can also be used as hosts.For example, mammalian trunk cell lines adapted to grow in suspension may be useful. Other examples of useful mammalian host cell lines include SV40 (COS-7) transformed monkey kidney CV1 cells, human embryonic kidney cell lines (e.g., 293 or 293 cells described in Graham et al., J. Gen Virol. 36:59 (1977)), baby hamster kidney cells (BHK), mouse Sertoli cells (e.g., TM4 cells described in Mather, Biol. Reprod. 23:243-251 (1980)), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical carcinoma cells (HELA), canine kidney cells (MDCK; buffalo rat liver cells (BRL 3A), human lung cells (W138), human liver cells (Hep G2), mouse mammary tumor (MMT 060562), e.g., Mather et al., Annals TRI cells, MRC 5 cells, and FS4 cells, described in NYAcad. Sci. 383:44-68 (1982). Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)), and myeloma cell lines, such as Y0, NS0, and Sp2 / 0. For a review of specific mammalian host cell lines suitable for antibody production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003).

[0144] Anti-CD3 antibodies and / or antigen-binding fragments thereof may be identified, screened, selected, or characterized for their physical / chemical properties and / or biological activity using various assays known in the art, such as ELISA and Western blotting. Alternatively, a competitive assay may be used to identify antibodies that compete with the anti-CD3 antibodies of the present disclosure for binding to CD3. In an example of a competitive assay, immobilized CD3 is incubated in a solution containing a first labeled antibody that binds to CD3 and a second unlabeled antibody that is tested for its ability to compete with the first antibody for binding to CD3. The second antibody may be present in hybridoma supernatant. As a control, immobilized CD3 is incubated in a solution containing the first labeled antibody but not the second unlabeled antibody. After incubation under conditions that allow the first antibody to bind to CD3, excess unbound antibody is removed, and the amount of label bound to the immobilized CD3 is measured. A significant decrease in the amount of label bound to immobilized CD3 in the test sample compared to the control sample indicates that the first and second antibodies compete for binding to CD3 (see, e.g., Harlow and Lane (1988) Antibodies: A Laboratory Manual, Ch. 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY)).

[0145] Biologically active anti-CD3 antibodies and / or antigen-binding fragments thereof may be identified using standard methods. Biological activity may include, for example, binding to CD3 on the surface of T cells, either in vivo, in vitro, or ex vivo. In the case of multispecific anti-CD3 antibodies (e.g., bispecific antibodies having one arm that binds to CD3 and another arm that binds to a different target, such as a tumor antigen or a cell surface antigen), biological activity may also include effector cell activation (e.g., activation of CD8+ T and / or CD4+ T cells), expansion of the effector cell population (i.e., an increase in the number of T cells), depletion of the target cell population (i.e., a decrease in the population of cells expressing a second biological molecule on their cell surface), and / or target cell killing.

[0146] Diagnostic and therapeutic uses of anti-CD3 antibodies and antigen-binding fragments thereof The anti-CD3 antibodies and / or antigen-binding fragments described herein may be used for diagnosis and / or detection. As used herein, "detection" includes quantitative or qualitative detection.

[0147] In some embodiments, a labeled anti-CD3 antibody is provided. The anti-CD3 antibodies and / or antigen-binding fragments thereof described herein may contain a label or moiety that is directly (e.g., fluorescent, dye, electron-dense, chemiluminescent, and radioactive) or indirectly (e.g., enzyme or ligand) detectable. Non-limiting examples of labels include radioisotopes such as 32P, 14C, 125I, 3H, and 131I; fluorophores such as rare earth chelates or fluorescein and its derivatives; rhodamine and its derivatives; dansyl; umbelliferone; luciferases such as firefly luciferase and bacterial luciferase (U.S. Pat. No. 4,737,456); luciferin; 2,3-dihydrophthalazinedione; horseradish peroxidase (HRP); alkaline phosphatase; β-galactosidase; and the like. Examples of suitable oxidases include oxidases, glucoamylases, lysozymes, monosaccharide oxidases such as glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, heterocyclic oxidases such as uricase and xanthine oxidase coupled with enzymes that use hydrogen peroxide to oxidize dye precursors such as HRP, lactoperoxidase, or microperoxidase, biotin / avidin, spin labels, bacteriophage labels, stable free radicals, and the like.

[0148] The CD3 antibodies and / or antigen-binding fragments thereof described herein, as well as pharmaceutical compositions of such antibodies, may be used in therapeutic methods. In one embodiment, the anti-CD3 antibodies and / or antigen-binding fragments thereof described herein, or pharmaceutical compositions containing such antibodies, may be used to treat or delay the progression of a cell proliferative disorder or an autoimmune disorder. In some embodiments, the anti-CD3 antibodies and antigen-binding fragments thereof may be used to treat cancer. Typically, tumor cells have an extracellular pH of about 6.3 to 6.5. The anti-CD3 antibodies and antigen-binding fragments described herein promote preferential CD3 binding at low pH values, such as about pH 6, thereby promoting binding and activity in and around the tumor microenvironment. In some embodiments, the use of anti-CD3 antibodies and antigen-binding fragments thereof may result in selective and sustained cytotoxic activity at or around the tumor site, thereby reducing or eliminating off-target effects.

[0149] "Disorder" refers to any condition or disease that would benefit from treatment, including, but not limited to, chronic and acute disorders or diseases, including pathological conditions that predispose the mammal to the disorder in question.

[0150] The terms "cell proliferative disorder" and "proliferative disorder" refer to disorders associated with some degree of abnormal cell proliferation. Cell proliferative disorders include cancers, e.g., tumors.

[0151] 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.

[0152] "Cancer" refers to the physiological condition in mammals characterized by uncontrolled cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies, and more specific examples include squamous cell carcinoma (e.g., squamous cell carcinoma), lung cancer, including small cell lung cancer, non-small cell lung cancer, lung adenoma and lung squamous cell carcinoma, cancer of the peritoneum, hepatocellular carcinoma, gastric cancer (including gastrointestinal cancer and gastrointestinal stromal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, cancer of the urinary tract, liver cancer (hepatoma), breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer (kidney cancer or renal cancer), and the like. cancer), prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, melanoma, superficial spreading melanoma, lentigo maligna melanoma, acral lentigo melanoma, nodular melanoma, multiple myeloma and B-cell lymphoma (low-grade / follicular non-Hodgkin's lymphoma (NHL), small lymphocytic (SL) NHL, intermediate-grade / follicular NHL, intermediate-grade diffuse NHL, high-grade immunoblastic NHL, high-grade lymphoblastic NHL, high-grade small noncleaved cell NHL) Cancers suitable for treatment with the antibodies of the present disclosure include breast cancer, colorectal cancer, rectal cancer, non-small cell lung cancer, glioblastoma, non-Hodgkin's lymphoma (NHL), renal cell carcinoma, prostate cancer, liver cancer, pancreatic cancer, soft tissue sarcoma, Kaposi's sarcoma, carcinoid carcinoma, head and neck cancer, ovarian cancer, mesothelioma, and multiple myeloma. In some embodiments, the cancer is selected from small cell lung cancer, glioblastoma, neuroblastoma, melanoma, breast cancer, gastric cancer, colorectal cancer (CRC), and hepatocellular carcinoma, although in some embodiments, the cancer is selected from non-small cell lung cancer, colorectal cancer, glioblastoma, and breast cancer, including metastatic forms of those cancers.In other embodiments, the cancer excludes Hodgkin's lymphoma, but is selected from the group consisting of germinal center B-cell-like (GCB) DLBCL, activated B-cell-like (ABC) DLBCL, follicular lymphoma (FL), mantle cell lymphoma (MCL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), marginal zone lymphoma (MZL), small lymphocytic leukemia (SLL), lymphoplasmacytic lymphoma (LL), Waldenstrom's macroglobulinemia (WM), and leukemia-associated lymphoma (LEM). Central nervous system lymphoma (CNSL), Burkitt lymphoma (BL), B-cell prolymphocytic leukemia, splenic marginal zone lymphoma, hairy cell leukemia, splenic lymphoma / leukemia, unclassifiable, splenic diffuse red pulp small B-cell lymphoma, hairy cell leukemia variant, Waldenstrom's macroglobulinemia, heavy chain disease, alpha heavy chain disease, gamma heavy chain disease, mucous membrane-associated lymphoid tissue disease, plasma cell myeloma, isolated plasmacytoma of bone, extraskeletal plasmacytoma, extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue Zone lymphoma (MALT lymphoma), nodal marginal zone lymphoma, pediatric nodal marginal zone lymphoma, pediatric follicular lymphoma, primary cutaneous follicle-centered lymphoma, T-cell / histiocytic-rich large B-cell lymphoma, primary DLBCL of the CNS, primary cutaneous DLBCL, foot type, EBV-positive DLBCL in the elderly, DLBCL associated with chronic inflammation, lymphomatoid granulomatosis, primary mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, AL Selected from K-positive large B-cell lymphoma, plasmablastic lymphoma, large B-cell lymphoma arising in HHV8-associated multicentric Castleman disease, a class of mature B-cell carcinomas including primary effusion lymphoma, B-cell lymphoma unclassifiable with features intermediate between diffuse large B-cell lymphoma and Burkitt lymphoma, and B-cell lymphoma unclassifiable with features intermediate between diffuse large B-cell lymphoma and classical Hodgkin lymphoma.

[0153] As used herein, "treatment" or "treat" or "treating" refers to clinical intervention in an attempt to alter the natural course of the individual being treated, and can be performed either for preventative purposes or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, prevention of disease onset or recurrence, alleviation of symptoms, reduction of any direct or indirect pathological consequences of the disease, prevention of metastasis, reduction in the rate of disease progression, improvement or palliation of the disease state, and remission or improved prognosis.

[0154] As used herein, the terms "prevent," "preventing," and "prevention" refer to the prevention or inhibition of the development or occurrence of a disorder or disease.

[0155] As used herein, the terms "amelioration" and "alleviation" refer to a decrease or lessening in the severity of a condition or any of its symptoms.

[0156] In some embodiments, the antibodies of the present disclosure are used to delay the onset of a disorder or disease or to slow the progression of a disorder or disease. As used herein, "delaying the progression" of a disorder or disease means postponing, preventing, slowing, inhibiting, stabilizing, and / or postponing the onset of the disorder or disease (e.g., a cell proliferative disorder, e.g., cancer). The delay can be for varying lengths of time, depending on the history of the disease and / or the individual being treated.

[0157] An effective amount of the antibody or composition may be administered to an individual suffering from cancer, or arthritis, rheumatoid arthritis, colitis, inflammatory bowel disease, autoimmune type 1 diabetes, etc. For example, an "effective amount" of an anti-CD3 antibody disclosed herein, or a composition (e.g., a pharmaceutical composition) comprising the antibody, is at least the minimum amount required to achieve a desired therapeutic or prophylactic result, such as a measurable improvement or prevention of a particular disorder, e.g., a cell proliferative disorder such as cancer, preferably without or with minimal toxic or adverse consequences. The effective amount may vary depending, inter alia, on the patient's disease state, age, sex, and weight, and the ability of the antibody (or antigen-binding fragment thereof) to elicit a desired response in the individual, and in some cases, by co-administration with one or more additional therapeutic agents.

[0158] In some embodiments, the anti-CD3 antibodies and / or antigen-binding fragments thereof described herein may be used to enhance immune function in individuals with cell proliferative or autoimmune disorders. Following administration, the antibodies or compositions may enhance immune function in individuals with cell proliferative or autoimmune disorders by activating effector cells (e.g., T cells, such as CD8+ and / or CD4+ T cells, including Tregs), expanding (increasing) the effector cell population, depleting the target cell population (e.g., cells expressing a second biological molecule recognized by an anti-CD3 antibody, such as a bispecific antibody of the invention), and / or killing target cells (e.g., target tumor cells).

[0159] The anti-CD3 antibodies and / or antigen-binding fragments thereof disclosed herein can be used to treat disorders including, but not limited to, proliferative disorders, oncological disorders, immuno-oncological disorders, neurological disorders, cognitive disorders, neurodegenerative disorders, and autoimmune disorders. In one embodiment, an effective amount of the anti-CD3 antibodies, alone or in combination with at least one additional agent, can be administered to an individual with such a disorder. Such an "individual" can be a mammal, particularly a human.

[0160] Non-limiting examples of additional therapeutic agents include chemotherapeutic agents, antibody-drug conjugates (ADCs), and / or biological modifiers. The chemotherapeutic agent may be selected from cyclophosphamide, doxorubicin, vincristine, and prednisolone (CHOP). The ADC may be selected from an anti-CD79b antibody-drug conjugate (e.g., CD79b-MC-vc-PAB-MMAE or an anti-CD79b antibody-drug conjugate described in any one of U.S. Patent No. 8,088,378 and / or US 2014 / 0030280, or polatuzumab vedotin), an anti-CD19 antibody-drug conjugate, an anti-CD22 antibody-drug conjugate, an anti-CD45 antibody-drug conjugate, and an anti-CD32 antibody-drug conjugate.Biological modifiers include BCL-2 inhibitors (e.g., GDC-0199 / ABT-199), lenalidomide (Revlimid®), PI3K-delta inhibitors (e.g., idelalisib (Zydelig®)), PD-1 axis binding antagonists, such as CD40, CD226, CD28, OX40 (e.g., AgonOX), GITR, CD137 (TNFRSF9, 4-1 agonists, such as agonistic antibodies directed against activating costimulatory molecules such as CD27 (also known as BB or ILA), CD27 (e.g., CDX-1127), HVEM or CD127; antagonists, such as antagonist antibodies directed against inhibitory costimulatory molecules such as CTLA-4 (also known as CD152), PD-1, TIM-3, BTLA, VISTA, LAG-3, B7-H3, B7-H4, IDO (e.g., 1-methyl-D-tryptophan (also known as 1-D-MT)), TIGIT, MICA / B, GITR (e.g., TRX518) or arginase; ipilimumab (also known as MDX-010, MDX-101, or Yervoy®), tremelimumab (also known as ticilimumab or CP-675,206, urelumab (also known as BMS-663513), MGA271, antagonists directed against TGF beta, such as, for example, metelimumab (also known as CAT-192), fresolimumab (also known as GC1008), LY2157299k, and adoptive transfer of T cells (e.g., cytotoxic T cells or CTLs) expressing a chimeric antigen receptor (CAR), for example, adoptive transfer of T cells comprising a dominant negative TGF beta receptor, such as a dominant negative TGF beta type II receptor.

[0161] The anti-CD3 antibodies and / or antigen-binding fragments thereof disclosed herein can be used to enhance immune function in individuals, e.g., humans, with such disorders. In one embodiment, a method of enhancing immune function comprises administering to the individual an amount of an anti-CD3 antibody effective to activate effector cells (e.g., T cells, e.g., CD8+ T cells and / or CD4+ T cells), expand (increase) the effector cell population, deplete the target cell population, and / or kill target cells (e.g., target tumor cells).

[0162] In a further aspect, pharmaceutical formulations are also provided that include the anti-CD3 antibodies and / or antigen-binding fragments described herein, e.g., for use in any of the above-described methods of treatment. A "pharmaceutical formulation" refers to a preparation in a form that allows the biological activity of an active ingredient contained therein, such as an anti-CD3 antibody described herein, to be effective, and preferably does not contain additional ingredients that are unacceptably toxic to the subject to which the formulation is administered.

[0163] In one embodiment, a pharmaceutical formulation comprises any of the anti-CD3 antibodies disclosed herein and a pharmaceutically acceptable carrier. A "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation, other than the active ingredient, that is not toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives. In another embodiment, a pharmaceutical formulation comprises any of the anti-CD3 antibodies provided herein and at least one additional therapeutic agent.

[0164] The antibodies of the present disclosure may be used alone or in combination with other agents in a treatment, for example, an anti-CD3 antibody and / or antigen-binding fragment thereof may be co-administered with at least one additional therapeutic agent. In certain embodiments, the additional therapeutic agent is a chemotherapeutic agent, a growth inhibitory agent, a cytotoxic agent, an agent used in radiation therapy, an anti-angiogenic agent, an apoptotic agent, an anti-tubulin agent, or other agent, such as an epidermal growth factor receptor (EGFR) antagonist (e.g., a tyrosine kinase inhibitor), a HER1 / EGFR inhibitor (e.g., erlotinib (Tarceva™)), a platelet-derived growth factor inhibitor (e.g., Gleevec™ (imatinib mesylate)), a COX-2 inhibitor (e.g., celecoxib), an interferon, a cytokine, an antibody other than the anti-CD3 antibodies of the disclosure, such as an antibody that binds to one or more of the following targets: ErbB3, ErbB4, PDGFR-beta, BIyS, APRIL, BCMA VEGF or VEGF receptor, TRAIL / Apo2, PD-1, PD-L1, PD-L2, or other bioactive or organic chemical agent.

[0165] In some embodiments, the present disclosure provides methods wherein the additional therapeutic agent is a glucocorticoid. In one embodiment, the glucocorticoid is dexamethasone.

[0166] Such combination therapy encompasses mixed administration (two or more therapeutic agents in the same formulation or in separate formulations) and separate administration, where administration of an antibody of the present disclosure can occur before, simultaneously with, and / or after administration of the additional therapeutic agent. In one embodiment, administration of an anti-CD3 antibody and administration of an additional therapeutic agent occur within about one month, or within about one, two, or three weeks, or within about one, two, three, four, five, or six days of each other. Anti-CD3 antibodies of the present disclosure (e.g., bispecific anti-CD3 antibodies of the present disclosure that bind to CD3 and a second biological molecule, e.g., a cell surface antigen such as a tumor antigen, e.g., a TDB antibody or variant thereof of the present disclosure) can also be used in combination with radiation therapy.

[0167] The disclosed antibodies (and / or any additional therapeutic agents) can be administered by any suitable means, including parenteral, intrapulmonary, and intranasal administration, or, if localized treatment is desired, by intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. In some embodiments, the antibody is administered subcutaneously. In some embodiments, an anti-CD3 antibody administered by subcutaneous injection exhibits a lower toxic response in patients than the same anti-CD3 antibody administered by intravenous injection. Dosing can be by any suitable route, e.g., by injection, e.g., intravenous or subcutaneous injection, depending in part on whether the administration is temporary or chronic. Various dosing schedules are contemplated herein, including, but not limited to, a single dose or multiple doses over various time points, a bolus dose, and pulse infusion.

[0168] The antibodies of the present disclosure are formulated, dosed, and administered in a manner consistent with sound medical practice. Factors to consider in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the administration schedule, and other factors known to medical professionals. Although not required, the antibodies may optionally be formulated with one or more agents currently used to prevent or treat the disorder in question. The effective amount of such other agents will depend on the amount of antibody present in the formulation, the type of disorder or treatment, and other factors discussed above. These will generally be used at the same doses and by the same routes of administration as those described herein, or at approximately 1-99% of the doses described herein, or at any dose and by any route empirically / clinically determined to be appropriate.

[0169] The appropriate dose of the disclosed antibodies (whether used alone or in combination with one or more other additional therapeutic agents) for the prevention or treatment of disease will depend on the type of disease being treated, the type of antibody, the severity and course of the disease, whether the antibody is being administered for prophylactic or therapeutic purposes, previous treatments, the patient's medical history and responsiveness to the antibody, and the discretion of the attending physician. The antibody is suitably administered to the patient at one time or over a series of treatments.

[0170] As a general rule, a therapeutically effective amount of an anti-CD3 antibody administered to a human, whether administered as a single dose or multiple doses, will be in the range of about 0.01 to about 100 mg / kg of patient body weight. In some embodiments, the antibody used is administered daily at, for example, about 0.01 to about 45 mg / kg, about 0.01 to about 40 mg / kg, about 0.01 to about 35 mg / kg, about 0.01 to about 30 mg / kg, about 0.01 to about 25 mg / kg, about 0.01 to about 20 mg / kg, about 0.01 to about 15 mg / kg, about 0.01 to about 10 mg / kg, about 0.01 to about 5 mg / kg, or about 0.01 to about 1 mg / kg. In one embodiment, an anti-CD3 antibody described herein is administered to a human at a dose of about 100 mg, about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, about 1000 mg, about 1100 mg, about 1200 mg, about 1300 mg, or about 1400 mg on day 1 of a 21-day cycle. The dose may be administered as a single dose or multiple doses (e.g., two or three doses), e.g., by infusion. When administered repeatedly over several days or longer, treatment is generally sustained until a desired suppression of disease symptoms occurs, depending on the condition. One exemplary dose of the antibody ranges from about 0.05 mg / kg to about 10 mg / kg. Thus, one or more doses of about 0.5 mg / kg, 2.0 mg / kg, 4.0 mg / kg, or 10 mg / kg (or any combination thereof) may be administered to the patient. Such doses may be administered intermittently, for example, weekly or every three weeks (e.g., the patient receives about two to about twenty, or, for example, about six, doses of anti-CD3 antibody). An initial higher loading dose may be administered, followed by one or more lower loading doses. The progress of this therapy is easily monitored by conventional techniques and assays.

[0171] In some embodiments, the disclosed methods may further comprise an additional therapy. The additional therapy may be radiation therapy, surgery, chemotherapy, gene therapy, DNA therapy, viral therapy, RNA therapy, immunotherapy, bone marrow transplant, nanotherapy, monoclonal antibody therapy, or a combination of the foregoing. The additional therapy may be in the form of adjuvant therapy or neoadjuvant therapy. In some embodiments, the additional therapy is administration of a small molecule enzyme inhibitor or an anti-metastatic agent. In some embodiments, the additional therapy is administration of a side effect-limiting agent (e.g., an agent intended to reduce the occurrence and / or severity of side effects of the therapy, such as an antiemetic). In some embodiments, the additional therapy is radiation therapy. In some embodiments, the additional therapy is surgery. In some embodiments, the additional therapy is a combination of radiation therapy and surgery. In some embodiments, the additional therapy is gamma irradiation. In some embodiments, the additional therapy may be the separate administration of one or more of the aforementioned therapeutic agents.

[0172] Another aspect of the present disclosure provides an article of manufacture containing materials useful for the treatment, prevention, and / or diagnosis of the aforementioned disorders. The article of manufacture includes a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc. The container may be formed from a variety of materials, such as glass or plastic. The container may hold the composition alone or in combination with another composition effective for the treatment, prevention, and / or diagnosis of a condition, and may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial with a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is an antibody of the present disclosure. The label or package insert indicates that the composition is used for treating the selected condition. Additionally, the article of manufacture may include (a) a first container containing a composition contained therein, the composition comprising an antibody of the present disclosure, and (b) a second container containing a composition contained therein, the composition comprising an additional cytotoxic agent or another therapeutic agent. The article of manufacture in this embodiment of the disclosure may further include a package insert indicating that the composition can be used to treat a particular condition. Alternatively, or in addition, the article of manufacture may further include a second (or third) container containing a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0173] Accordingly, the manufacture and / or preparation of pharmaceutical compositions comprising the anti-CD3 antibodies and / or antigen-binding fragments disclosed herein are also contemplated. The compositions may be used alone or in combination with other active agents to treat cell proliferative disorders (e.g., cancer) or autoimmune disorders (e.g., arthritis, rheumatoid arthritis, colitis, inflammatory bowel disease, autoimmune type 1 diabetes, etc.).

[0174] In some embodiments, pharmaceutical compositions comprising the anti-CD3 antibodies and / or antigen-binding fragments thereof described herein are prepared, for example, by combining the antibodies having the desired purity with one or more pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)) in the form of a lyophilized formulation or an aqueous solution, optionally prepared for modified (e.g., sustained) release. Exemplary lyophilized antibody formulations are described in U.S. Pat. No. 6,267,958. Aqueous antibody formulations include those described in U.S. Pat. No. 6,171,586 and WO 2006 / 044908, the latter of which contains a histidine-acetate buffer.

[0175] Pharmaceutically acceptable carriers are generally non-toxic to recipients at the dosages and concentrations employed and include, but are not limited to, buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl alcohol or benzyl alcohol, alkyl parabens such as methyl paraben or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol); low molecular weight (less than about 10 residues) polypeptides; Examples of pharmaceutically acceptable carriers include proteins such as serum albumin, gelatin, or immunoglobulins, hydrophilic polymers such as polyvinylpyrrolidone, amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine, monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin, chelating agents such as EDTA, sugars such as sucrose, mannitol, trehalose, or sorbitol, salt-forming counterions such as sodium, metal complexes (e.g., Zn-protein complexes), and / or non-ionic surfactants such as polyethylene glycol (PEG). Pharmaceutically acceptable carriers herein also include interstitial drug dispersion agents, such as soluble neutral-active hyaluronidase glycoprotein (sHASEGP), for example, human soluble PH-20 hyaluronidase glycoprotein, for example, rHuPH20 (HYLENEX®, Baxter International, Inc.). Certain exemplary sHASEGPs, including rHuPH20, and methods of using same are described in U.S. Patent Application Publications 2005 / 0260186 and 2006 / 0104968.

[0176] Such formulations may contain more than one active ingredient, if necessary for the particular indication being treated, preferably ingredients with complementary activities that do not adversely affect each other, and that are present in amounts that are effective for the purpose intended. For example, it may be desirable to further provide additional therapeutic agents (e.g., chemotherapeutic agents, cytotoxic agents, growth inhibitory agents, and / or antihormonal agents, etc.).

[0177] The active ingredient may be encapsulated in microcapsules prepared by droplet formation techniques or by interfacial polymerization, such as hydroxymethylcellulose or gelatin microcapsules, and poly(methyl methacrylate) microcapsules, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or in the form of macroemulsions, respectively. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980). [Example]

[0178] Example 1: Construction of an engineered pH-dependent CD3 library The combinatorial histidine substitution library was derived from the parent anti-CD3 antibody clone ADI-26906 (Antibody No. 1 in Table 1). ADI-26906 was first disclosed in PCT / US2018 / 031705 (ADI-26906 was not pH engineered), which is incorporated herein by reference in its entirety. The following three library designs were used to incorporate histidine: 1) H3+L3 jumping double with L1 single or double histidine (His) substitution (with or without an NNK polymorphism adjacent to the His), thereby generating 3.4 x 10 5 2) adding the H3 jumping doublet to a pre-generated H1 / H2 diversity library to generate a theoretical diversity of 6.8 × 10 8and 3) walking singlets of H3+L3 NNK / His or His / NNK, resulting in a theoretical diversity of 1.2 × 10 5 This yields a theoretical diversity of 10 ...

[0179] Five selection rounds were performed using three libraries against biotinylated CD3 antigen. For the first selection round of the H1 / H2+H3 library, magnetic bead sorting using the Miltenyi MACS system was performed essentially as described (Siegel et al., J Immunol Methods. 2004 Mar;286(1-2):141-53). Briefly, approximately 10 9Yeast cells were incubated with 1 mL of 100 nM biotinylated CD3 antigen in FACS wash buffer (PBS, pH 6.0, containing 0.1% BSA) at room temperature for 15 minutes. After washing once with 50 mL of ice-cold wash buffer, the cell pellet was resuspended in 40 mL of wash buffer, and 500 μl of Streptavidin MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany, catalog number 130-048-101) was added to the yeast and incubated at 4°C for 15 minutes. The yeast were then pelleted, resuspended in 5 mL of wash buffer, and loaded onto a MACS LS column (Miltenyi Biotec, Bergisch Gladbach, Germany, catalog number 130-042-401). After loading 5 mL, the column was washed three times with 3 mL of FACS wash buffer. The column was then removed from the magnetic field, and the yeast were eluted with 5 mL of growth medium and then grown overnight. For the two low diversity libraries, a first round of selection was performed using flow cytometry (FACS). Briefly, yeast cells (approximately 10 9 Yeast cells (10 ...

[0180] Following the first round of MACS or FACS, four rounds of sorting were performed using FACS and pH toggle selection methods (see Figure 1).

[0181] Purified CD3 protein antigen was biotinylated using the EZ-Link Sulfo-NHS-Biotinylation Kit (Thermo Scientific). The CD3 antigen was concentrated to approximately 1 mg / mL and buffer-exchanged into PBS, followed by the addition of a 1:7.5 molar ratio of biotinylation reagent (EZ-Link Sulfo-NHS-Biotinylation Kit, Thermo Scientific, catalog number 21425). The mixture was kept overnight at 4°C and further buffer-exchanged to remove free biotin in the solution. Biotinylation was confirmed via streptavidin sensor binding of the labeled protein on a ForteBio. Successful biotinylation of the CD3 protein antigen was confirmed via detectable binding to a streptavidin-coupled biosensor installed in a ForteBio Octet™ Red384 Interferometer (Pall ForteBio, Menlo Park, CA) according to the manufacturer's guidelines (data not shown). In CD3 presaturation method #1 (shown in Figure 1A), yeast cells were presaturated with native (non-biotinylated) CD3 antigen at pH 7.4 for 10 minutes. Next, the yeast cells were washed at pH 7.4 and incubated in medium at pH 6.0 for 10 minutes to dissociate the antigen. Control cells were washed and incubated at pH 7.4. Finally, yeast cells were incubated with biotinylated CD3 antigen (shown as a green circle with a star in Figure 1A) at pH 6 for 10 minutes. Control cells were incubated with biotinylated CD3 antigen at pH 7.4. The labeled binders were then sorted and characterized at pH 6. In CD3 presaturation method #2 (shown in Figure 1B), yeast cells were presaturated with native CD3 antigen at pH 6.0 for 10 minutes. Next, the yeast cells were washed at pH 6.0 and incubated at either pH 7.4 or pH 6.0 for 10 minutes. Finally, yeast cells were incubated with biotinylated CD3 antigen for 10 min at the opposite pH (if cells had been incubated at pH 6.0 in the previous step, they were incubated at pH 7.4; conversely, cells incubated at pH 7.4 in the previous step were incubated at pH 6.0).Binders labeled with biotinylated CD3 antigen were then sorted and characterized.

[0182] Three libraries from the MACS / FACS selection were subjected to four rounds of FACS selection, with approximately 1 x 10 per library. 8 Yeast cells were pelleted, washed three times with wash buffer, and incubated with 100 nM biotinylated CD3 antigen at pH 6.0 or pH 7.4 for at least 10 minutes at room temperature, separately, or through the presaturation and pH toggle procedure described in Method #2. Yeast cells were then washed twice and stained with goat anti-human F(ab')2 kappa-FITC (Southern Biotech, Birmingham, AL, Catalog #2062-02) diluted 1:100 and either streptavidin-Alexa Fluor 633 (Life Technologies, Grand Island, NY, Catalog #S21375) diluted 1:500 or extravidin-phycoerythrin (Sigma-Aldrich, St. Louis, CA, Catalog #E4011) diluted 1:50 for 15 minutes at 4°C. After washing twice with ice-cold wash buffer, the cell pellet was resuspended in 0.4 mL of wash buffer and transferred to a sort tube capped with a strainer. Sorting was performed using a FACS ARIA sorter (BD Biosciences), and sorting gates were determined to select either CD3 binders at pH 6 or non-binders at pH 7.4. The selected population from the first round of FACS was advanced to the next round.

[0183] The second, third, and fourth FACS rounds for the above-selected population included positive sorting for CD3 binders at pH 6.0 and negative sorting to reduce pH 7.4 binders and polyspecific reagent binders (Xu et al., Protein Eng Des Sel. 2013 Oct;26(10):663-70). In the second round of FACS (R3), cells were processed through method #2 presaturation and pH toggle (described above) or negatively sorted for nonbinders selected at pH 7.4. In the third round of FACS (R4), the output from R3 was pooled with the output from R2 CD3 presaturation method #2, and CD3 presaturation method #1 was performed. In the final round of FACS (R5), the output from R4 was confirmed for PSR reactivity and human and cynomolgus monkey (Cyno) CD3 binding at pH 6 and 7.4. The output of each round was plated and isolates were selected for sequencing and characterization.

[0184] Figure 2 shows exemplary FACS plots of round 1 and round 2 selections from one library. Similar binding profiles were observed for all libraries. Briefly, during round 1, cells were positively sorted using 100 nM human CD3εδ heterodimer (HuCD3-hd) at pH 6. During round 2, cells were positively sorted using 100 nM HuCD3-hd at pH 6.0 and negatively sorted using 100 nM HuCD3-hd at pH 7.4, or presaturated using method #2 described above. Binding to cynomolgus monkey CD3 (CyCD3-hd) at pH 6.0 was also confirmed. Arrows indicate sorted cells carried over to the next sorting round.

[0185] Figure 3 shows an exemplary FACS plot from round 3, comparing the input of a pH 6.0 positive sort from round 2 with a pH 7.4 negative sort. Briefly, the sort from round 2 was incubated with 100 nM HuCD3-hd at pH 6.0 and pH 7.4. The overlay column shows that the input cell population (from the round 2 sort) exhibits higher binding at pH 6.0 compared to pH 7.4. Cells were carried forward to the next selection round using the presaturation / toggle method #2.

[0186] Figure 4 shows exemplary FACS plots from rounds 4 and 5. Round 4 compared cells incubated with 100 nM HuCD3-hd at pH 6 and pH 7.4. Round 4 also compared cells pre-saturated / toggled at pH 6 and pH 7.4. Round 5 compared cells incubated with either 100 nM HuCD3-hd or 100 nM CyCD3-hd at pH 6 (red) and pH 7.4 (gray).

[0187] Example 2: Determination of the affinity of anti-CD3 antibodies to CD3 The affinity of anti-CD3 antibodies for CD3 at pH 6.0 and pH 7.4 was measured on the ForteBio Octet using a rate constant (k a , k d , K. D) were determined. ForteBio affinity measurements were performed generally as previously reported (Estep et al., MAbs. 2013 5(2):270-8). Briefly, ForteBio affinity measurements were performed by online loading of antibody (IgG) onto the AHC sensor. The sensor was equilibrated offline in assay buffer for 30 minutes and then monitored online for 60 seconds to establish a baseline. For avidity binding measurements, 100 nM of antigen (human or cynomolgus monkey CD3) was exposed to the IgG-loaded sensor for 3 minutes, after which they were transferred to assay buffer for 3 minutes and dissociation rate measurements were performed. Rate data were fitted using a 1:1 binding model in the data analysis software provided by ForteBio. Table 2 presents the rate constants of selected clones. Table 3 shows the equilibrium dissociation constants (K) of selected clones to human CD3 at pH 6.0 and 7.4. D ) is presented.

[0188] The specificity of anti-CD3 antibodies for human CD3+ Jurkat cells compared to CHO-S cells at pH 6.0 and 7.4 was determined using a FACS cell-binding assay. Briefly, CD3+ human Jurkat cells and CHO-S cells were lysed and washed with cold PBSF buffer, pH 7.4 (PBS + 0.1% BSA, pH 7.4). Approximately 200,000 cells were dispensed per well of a 96-well plate and pelleted by centrifugation (500 x g for 5 minutes). Cells were washed with either PBSF pH 7.4 or PBSF pH 6.0 (PBS + 0.1% BSA, pH 6.0) and then resuspended in 100 μl of either PBSF pH 7.4 or PBSF pH 6.0 containing yeast-produced IgG antibody (100 nM). The mixture (cells + antibody) was incubated on ice for 20 minutes and then washed twice with either PBSF pH 7.4 or PBSF pH 6.0. Cells were resuspended in 50 μl of propidium iodide (1:500 dilution) and anti-human IgG-RPE (1:100 dilution) prepared in either PBSF pH 7.4 or PBSF pH 6.0. After 20 minutes of incubation on ice in the dark, cells were washed twice with either PBSF pH 7.4 or PBSF pH 6.0. Binding was analyzed using a FACS Canto II. The mean fluorescence intensity (MFI) of selected clones at pH 6.0 and 7.4 is shown in Table 3.

[0189] Figure 5A shows the HuCD3 binding response at pH 6 (x-axis) compared to the HuCD3 binding response at pH 7.4 (y-axis) for 236 unique clones from the output of round 2 / 3 sorting. Figure 5B shows the KD values ​​of HuCD3 at pH 6 (x-axis) compared to the KD values ​​of HuCD3 at pH 7.4 (y-axis) for 236 unique clones from the output of round 2 / 3 sorting. Blue circles represent round 2 / 3 clones obtained via presaturation / toggle sorting at pH 6.0, yellow circles represent round 2 / 3 clones obtained via negative sorting at pH 7.4, and red circles represent the parent clone ADI-26906. The results show that negative sorting at pH 7.4 in round 2 / 3 tends to yield more pH-selective binders, but the response or affinity at pH 6.0 was weak, designated as group 2 binders. Positive selection at pH 6.0 and presaturation / toggle sorting yielded clones with mixed selectivity but high response / affinity, which were designated as group 1 binders.

[0190] Group 1 binders can include, for example, ADI-48592 (Ab125), ADI-48595 (Ab178), ADI-48650 (Ab77), ADI-48652 (Ab81), ADI-48662 (Ab116), and ADI-48666 (Ab177). Examples of group 2 binders include ADI-48588 (Ab58), ADI-48587 (Ab36), ADI-48577 (Ab193), ADI-48590 (Ab91), ADI-48581 (Ab237), ADI-48575 (Ab113), ADI-48593 (Ab158), ADI-48591 (Ab102), ADI-48647 (Ab65), ADI-48636 (Ab230), and ADI ADI-48586 (Ab25), ADI-48646 (Ab53), ADI-48638 (Ab22), ADI-48597 (Ab180), ADI-48601 (Ab191), ADI-48576 (Ab182), ADI-48643 (Ab46), ADI-48624 (Ab241), ADI-48632 (Ab15), ADI-48635 (Ab17), and ADI-48645 (Ab49).

[0191] Figure 6 shows exemplary kinetics from a ForteBio experiment for four clones compared to the parent clone ADI-26906. D was calculated at pH 7.4 and pH 6.0. D The ratio of K at pH 7.4 D pH 6.0 K D The example shows that some clones designated as Group 1 binders, such as SAD10318_P02_A05 (ADI-48595) and SAD10318_P02_C04 (ADI-48592), exhibited stronger (lower K) at pH 6.0 compared to pH 7.4. DThe results show that the clones bound at pH 6.0 (at pH 7.4). For example, some clones designated as group 2 binders, such as SAD10318_P01_A03 (ADI-48587) and SAD10318_P01_E01 (ADI-48577), were non-binders at pH 7.4 but bound at pH 6.0. Amino acid substitutions in the CDRH3, CDRL1, and CDRL3 regions that may account for differences in binding are highlighted in the sequence column of Figure 6 (SEQ ID NOS: 576-590, in order of appearance). Table 2 provides additional kinetic and PSR data for selected clones. For example, clones such as ADI-48576, ADI-48577, ADI-48587, ADI-48592, ADI-48595, ADI-48635, ADI-48650, ADI-48652, ADI-48666, ADI-48643, and ADI-48645 exhibit pH-dependent binding (stronger binding at pH 6.0 compared to binding at pH 7.4), low PSR scores, and offer a broad range of affinity for CD3.

[0192] Analysis of 258 unique clones identified using the methods of the present disclosure revealed a consensus motif within the CDR regions. In some embodiments, the present disclosure provides antibodies comprising a CDRH3-binding domain comprising a consensus motif, the consensus motif comprising the sequence AX1DX2YX3HX4FYDV, where X1 is R or H, X2 is A or H, X3 is G, H or P, and X4 is Y, H, D, V, E, S, N, L, M, I, G, A, Q, or T (SEQ ID NO: 1). In some embodiments, at least one of X1, X2, X3, and X4 is H. The following 120 clones contain the following sequence motifs: SAD10318_P01_A02; SAD10318_P01_G02; SAD10318_P01_D03; SAD10318_P01_G03; SAD10318_P01_H03; SAD10318_P02_D05; SAD10318_P02_H05; SAD10318_P02_G06; SAD10318_P03_ C08;SAD10318_P03_H08;SAD10318_P03_G09;SAD10318_P04_H10;SAD10318_P04_D11;SAD10319_P01_A01 ;SAD10319_P01_C01;SAD10319_P01_E01;SAD10319_P01_A02;SAD10319_P01_C02;SAD10319_P01_F02;SAD 10319_P01_H02;SAD10319_P01_B03;SAD10319_P01_C03;SAD10319_P01_D03;SAD10319_P01_F03;SAD103 19_P02_A04;SAD10319_P02_C04;SAD10319_P02_E04;SAD10319_P02_F04;SAD10319_P02_A05;SAD10319_P 02_B05;SAD10319_P02_C05;SAD10319_P02_G05;SAD10319_P02_A06;SAD10319_P02_B06;SAD10319_P02_ C06;SAD10319_P02_D06;SAD10319_P02_F06;SAD10319_P02_G06;SAD10319_P03_C07;SAD10319_P03_H07;SAD10319_P03_D08;SAD10319_P03_E08;SAD10319_P03_E09;SAD10319_P03_F09;SAD10319_P04_A10;SAD10319_P04_G10;SAD10319_P04_E11;SAD10319_P04_F11;SAD10319_P04_G11;SAD10319_P04_C12;SAD10319_P04_D12;SAD10320_P01_B01;SAD10320_P01_D01;SAD10320_P01_E01;SAD10320_P01_G01;SAD10320_P01_H01;SAD10320_P01_A02;SAD10320_P01_F02;SAD10320_P01_G02;SAD10320_P01_H02;SAD10320_P01_C03;SAD10320_P01_D03;SAD10320_P01_E03;SAD10320_P01_F03;SAD10320_P01_G03;SAD10320_P02_A04;SAD10320_P02_B04;SAD10320_P02_E04;SAD10320_P02_H04;SAD10320_P02_A05;SAD10320_P02_B05;SAD10320_P02_C05;SAD10320_P02_B06;SAD10320_P02_D06;SAD10320_P02_E06;SAD10320_P03_B07;SAD10320_P03_H07;SAD10320_P03_C08;SAD10320_P03_D08;SAD10320_P03_F08;SAD10320_P03_H08;SAD10320_P03_A09;SAD10320_P03_C09;SAD10320_P03_D09;SAD10320_P03_F09;SAD10320_P04_A10;SAD10320_P04_C10;SAD10320_P04_D10;SAD10320_P04_E10;SAD10320_P04_G10;SAD10320_P04_D11;SAD10320_P04_E11;SAD10320_P04_F11;SAD10320_P04_H11;SAD10320_P04_A12;SAD10320_P04_D12;SAD10320_P04_E12;SAD10320_P04_F12;SAD10319_P05_A01;SAD10319_P05_A05;SAD10319_P05_B02;SAD10319_P05_C01;SAD10319_P05_C03;SAD10319_ P05_C05;SAD10319_P05_D02;SAD10319_P05_D03;SAD10319_P05_D05;SAD10319_P05_E04;SAD10319_P05_F01;SA D10319_P06_B10;SAD10319_P06_B11;SAD10319_P06_C10;SAD10319_P06_C12;SAD10319_P06_E08;SAD10319_P06_F07;SAD10319_P06_F10;SAD10319_P06_G09;SAD10319_P06_H07;SAD10319_P06_H08;and SAD10319_P06_H10.;

[0193] In some embodiments, the disclosure provides antibodies comprising a CDRH3-binding domain comprising a consensus motif, the consensus motif comprising the sequence ARDX1YGX2X3X4YDX5, where X1 is A or H, X2 is R or H, X3 is H or Y, X4 is F or H, and X5 is H or V (SEQ ID NO: 2). In some embodiments, at least one of X1, X2, X3, X4, and X5 is H.The following 57 clones contain the following consensus motif: LAD5224_P03_A01; SAD10318_P01_B01; SAD10318_P01_F01; SAD10318_P02_B05; SAD10318_P02_F05; SAD10318_P02_G05; SAD10318_P03_B07; SAD10318_P03_G07; SAD10318_P03_A08; SAD10318_P03_A09; SAD10318_P04_E10; SAD10318_P04_E11; SAD10318_P04_H1 1;SAD10319_P01_D01;SAD10319_P01_F01;SAD10319_P01_G01;SAD10319_ P01_D02;SAD10319_P01_E02;SAD10319_P02_B04;SAD10319_P02_E05;SAD 10319_P02_E06;SAD10319_P02_H06;SAD10319_P03_G08;SAD10319_P03_B 09;SAD10319_P03_G09;SAD10319_P04_B10;SAD10319_P04_C11;SAD10319 _P04_D11;SAD10319_P04_F12;SAD10319_P04_H12;SAD10320_P01_E02;SA D10320_P02_C04;SAD10320_P02_C06;SAD10320_P02_G06;SAD10319_P05_ A02;SAD10319_P05_B03;SAD10319_P05_B04;SAD10319_P05_D01;SAD1031 9_P05_G02;SAD10319_P05_G03;SAD10319_P05_H06;SAD10319_P06_A07;S AD10319_P06_A10;SAD10319_P06_A11;SAD10319_P06_E09;SAD10319_P06_E10;SAD10319_P06_G11;SAD10319_P06_H11;LAD9953_P01_H01;LAD9954_P01_B02;LAD9955_P01_G02;LAD9956_P01_C03;LAD9959_P01_E04;LAD9960_P01_D05;LAD9963_P01_E06;LAD9964_P01_C07; and LAD9966_P01_A08.

[0194] In some embodiments, the disclosure provides antibodies comprising a CDRH3-binding domain comprising a consensus motif comprising the sequence ARDAHX1X2YX3X4DX5, where X1 is G, E, or R, X2 is R or H, X3 is F or H, X4 is Y or H, and X5 is V or H (SEQ ID NO: 3). In some embodiments, at least one of X1, X2, X3, X4, and X5 is H. The following 23 clones contain the following consensus motif: SAD10318_P01_G01; SAD10318_P01_F02; SAD10318_P01_C03; SAD10318_P01_E03; SAD10318_P01_F03; SAD10318_P02_B04; SAD10318_P02_D04; SAD10318_P02_D06; SAD10318_P03_F07; SAD10318_P04_F11; SAD10318_P04 _H12;SAD10319_P02_D04;SAD10319_P02_H04;SAD10319_P02_D05;SAD10319_P03_G07;SAD10319_P04_C10;SAD10319_P04_B11;SAD10319_P04_B12;SAD10320_P02_A06;SAD10319_P05_A03;SAD10319_P05_B05;SAD10319_P05_G04; and SAD10319_P06_D12.

[0195] In some embodiments, the disclosure provides an antibody comprising a CDRH3-binding domain comprising a consensus motif comprising the sequence ARDAX1HRX2FYDV, where X1 is H, Y, S, G, A, T, V, or R, and X2 is Y or H (SEQ ID NO: 4). In some embodiments, at least one of X1 and X2 is H. The following 19 clones contain this consensus motif: SAD10318_P01_E01; SAD10318_P01_H01; SAD10318_P01_D02; SAD10318_P02_C04; SAD10318_P02_C05; SAD10318_P02_B06; SAD10318_P02_E06; SAD10318_P03_D09; SAD10318_P04 _A12;SAD10319_P02_F05;SAD10319_P03_H08;SAD10320_P01_F01;SAD10320_P01_C02;SAD10320_P01_H03;SAD10320_P02_D05;SAD10320_P02_H05;SAD10320_P03_E07;SAD10320_P04_A11; and SAD10319_P05_G01.

[0196] In some embodiments, the disclosure provides antibodies comprising a CDRH3-binding domain comprising a consensus motif, the consensus motif comprising the sequence ARDX1YHRYFYDX2, where X1 is H or A, and X2 is H, V, or M (SEQ ID NO: 5). In some embodiments, at least one of X1 and X2 is H. The following 15 clones comprise the following consensus motif: SAD10318_P01_D01; SAD10318_P01_B02; SAD10318_P01_A03; SAD10318_P02_H04; SAD10318_P02_A05; SAD10318_P03_E07; SAD10318_P03 _B08;SAD10318_P03_D08;SAD10318_P03_E08;SAD10318_P03_F08;SAD10318_P03_G08;SAD10318_P03_C09;SAD10318_P04_B11;SAD10319_P01_H01;and SAD10319_P04_E12.

[0197] In some embodiments, the disclosure provides antibodies comprising a CDRH3-binding domain comprising a consensus motif comprising the sequence AX1DAYX2X3X4HX5DV, where Xi is R or H, X2 is G or H, X3 is H or R, X4 is N, F or Y, and X5 is Y or H (SEQ ID NO: 6). In some embodiments, at least one of Xi, X2, X3, X4, and X5 is H. The following 14 clones contain the consensus motif: SAD10318_P01_C01; SAD10318_P02_G04; SAD10318_P03_E09; SAD10318_P03_F09; SAD10318_P04_C10; SAD10318_P04_D10; SAD10318_P04_F10; SAD10318_P04_G11; SAD10318_P04_G12; SAD10320_P02_F05; SAD10320_P02_F06; SAD10320_P02_H06; SAD10320_P04_F10; and SAD10319_P05_D04.

[0198] In some embodiments, the disclosure provides antibodies comprising a CDRH3-binding domain comprising a consensus motif, the consensus motif comprising the sequence ARDX1X2GRYFYDV, where X1 is M, Q, or H, and X2 is R or H (SEQ ID NO: 7). In some embodiments, at least one of X1 and X2 is H. The following seven clones comprise the following sequence motif: SAD10318_P02_E04; SAD10318_P04_C11; SAD10318_P04_F12; SAD10319_P02_H05; SAD10320_P01_A03; SAD10320_P01_B03; and SAD10320_P02_E05.

[0199] In some embodiments, the disclosure provides antibodies comprising a CDRH3-binding domain comprising a consensus motif comprising the sequence ARDX1X2X3RYFYDX4, where X1 is H or A, X2 is T, Y, or H, X3 is G or H, and X4 is V or H (SEQ ID NO: 8). In some embodiments, at least one of X1, X2, X3, and X4 is H. The following clones contain the following sequence motifs: ADI-26906; ADI-48584; ADI-57317; ADI-57319; ADI-57323; ADI-57328; ADI-48639; ADI-57300; ADI-57333; ADI-57336; ADI-57337; ADI-48587; ADI-57343; ADI-48648; ADI-48650; ADI-48 589;ADI-48652;ADI-48654;ADI-48592;ADI-57401;ADI-57406;ADI-57274;ADI-57413;ADI-57414;ADI-5 7415;ADI-57416;ADI-57417;ADI-57275;ADI-57427;ADI-57428;ADI-57437;ADI-57438;ADI-48594;ADI-5 7439;ADI-57440;ADI-57441;ADI-57442;ADI-57443;ADI-57444;ADI-57445;ADI-48666;ADI-48595;ADI- 48597;ADI-48576;ADI-57277;ADI-57279;ADI-57280;ADI-57281;ADI-48601;ADI-48577;ADI-57284;ADI- 48604;ADI-48606;ADI-57285;ADI-48608;ADI-48609;ADI-48610;ADI-48614;ADI-48615;ADI-48617;ADI -57295;ADI-48580;ADI-48622;ADI-57299;ADI-57300;ADI-48623;ADI-57303;ADI-48582; and ADI-57311.

[0200] In some embodiments, the disclosure provides antibodies comprising a CDRH3-binding domain comprising a consensus motif, the consensus motif comprising the sequence ARDX1X2X3X4YFYDX5, where X1 is H or A, X2 is T, Y, or H, X3 is G or H, X4 is H, R, V, or I, and X5 is V or H (SEQ ID NO: 43). In some embodiments, at least one of X1, X2, X3, X4, and X5 is H. The following 11 clones contain the following consensus motif: ADI-48576; ADI-48577; ADI-48587; ADI-48592; ADI-48595; ADI-48635; ADI-48650; ADI-48652; ADI-48666; ADI-48643; and ADI-48645.

[0201] In some embodiments, the disclosure provides an antibody comprising a CD3-binding domain, CDRH3, wherein the CDRH3-binding domain comprises a consensus motif comprising the sequence AX1DX2X3X4X5X6X7X8DX9, where Xi is R or H, X2 is A, H, M, or Q, X3 is Y, H, S, G, A, T, V, or R, X4 is G, H, P, E, or R, X5 is H or R, X6 is Y, N, F, H, D, E, S, L, M, I, G, A, Q, or T, X7 is F or H, X8 is Y or H, and X9 is V, H, or M (SEQ ID NO: 58). In some embodiments, at least one of Xi, X2, X3, X4, X5, X6, X7, X8, and X9 is H. The following clones contain the following sequence motifs: SAD10318_P01_A02; SAD10318_P01_G02; SAD10318_P01_D03; SAD10318_P01_G03; SAD10318_P01_H03; SAD10318_P02_D05; SAD10318_P02_H05; SAD10318_P02_G06; SAD10318_P03_C08;SAD10318_P03_H08;SAD10318_P03_G09;SAD10318_P04_H10;SAD10318_P0 4_D11;SAD10319_P01_A01;SAD10319_P01_C01;SAD10319_P01_E01;SAD10319_P01_A02;SAD103 19_P01_C02;SAD10319_P01_F02;SAD10319_P01_H02;SAD10319_P01_B03;SAD10319_P01_C03; SAD10319_P01_D03;SAD10319_P01_F03;SAD10319_P02_A04;SAD10319_P02_C04;SAD10319_P0 2_E04;SAD10319_P02_F04;SAD10319_P02_A05;SAD10319_P02_B05;SAD10319_P02_C05;SAD10 319_P02_G05;SAD10319_P02_A06;SAD10319_P02_B06;SAD10319_P02_C06;SAD10319_P02_D06;SAD10319_P02_F06;SAD10319_P02_G06;SAD10319_P03_C07;SAD10319_P03_H07;SAD10319_P03_D08;SAD10319_P03_E08;SAD10319_P03_E09;SAD10319_P03_F09;SAD10319_P04_A10;SAD10319_P04_G10;SAD10319_P04_E11;SAD10319_P04_F11;SAD10319_P04_G11;SAD10319_P04_C12;SAD10319_P04_D12;SAD10320_P01_B01;SAD10320_P01_D01;SAD10320_P01_E01;SAD10320_P01_G01;SAD10320_P01_H01;SAD10320_P01_A02;SAD10320_P01_F02;SAD10320_P01_G02;SAD10320_P01_H02;SAD10320_P01_C03;SAD10320_P01_D03;SAD10320_P01_E03;SAD10320_P01_F03;SAD10320_P01_G03;SAD10320_P02_A04;SAD10320_P02_B04;SAD10320_P02_E04;SAD10320_P02_H04;SAD10320_P02_A05;SAD10320_P02_B05;SAD10320_P02_C05;SAD10320_P02_B06;SAD10320_P02_D06;SAD10320_P02_E06;SAD10320_P03_B07;SAD10320_P03_H07;SAD10320_P03_C08;SAD10320_P03_D08;SAD10320_P03_F08;SAD10320_P03_H08;SAD10320_P03_A09;SAD10320_P03_C09;SAD10320_P03_D09;SAD10320_P03_F09;SAD10320_P04_A10;SAD10320_P04_C10;SAD10320_P04_D10;SAD10320_P04_E10;SAD10320_P04_G10;SAD10320_P04_D11;SAD10320_P04_E11;SAD10320_P04_F11;SAD10320_P04_H11;SAD10320_P04_A12;SAD10320_P04_D12;SAD10320_P04_E12;SAD10320_P04_F12;SAD10319_P05_A01;SAD10319_P05_A05;SAD10319_P05_B02;SAD10319_P05_C01;SAD10319_P05_C03;SAD10319_P05_C05;SAD10319_P05_D02;SAD10319_P05_D03;SAD10319_P05_D05;SAD10319_P05_E04;SAD10319_P05_F01;SAD10319_P06_B10;SAD10319_P06_B11;SAD10319_P06_C10;SAD10319_P06_C12;SAD10319_P06_E08;SAD10319_P06_F07;SAD10319_P06_F10;SAD10319_P06_G09;SAD10319_P06_H07;SAD10319_P06_H08;SAD10319_P06_H10;LAD5224_P03_A01;SAD10318_P01_B01;SAD10318_P01_F01;SAD10318_P02_B05;SAD10318_P02_F05;SAD10318_P02_G05;SAD10318_P03_B07;SAD10318_P03_G07;SAD10318_P03_A08;SAD10318_P03_A09;SAD10318_P04_E10;SAD10318_P04_E11;SAD10318_P04_H11;SAD10319_P01_D01;SAD10319_P01_F01;SAD10319_P01_G01;SAD10319_P01_D02;SAD10319_P01_E02;SAD10319_P02_B04;SAD10319_P02_E05;SAD10319_P02_E06;SAD10319_P02_H06;SAD10319_P03_G08;SAD10319_P03_B09;SAD10319_P03_G09;SAD10319_P04_B10;SAD10319_P04_C11;SAD10319_P04_D11;SAD10319_P04_F12;SAD10319_P04_H12;SAD10320_P01_E02;SAD10320_P02_C04;SAD10320_P02_C06;SAD10320_P02_G06;SAD10319_P05_A02;SAD10319_P05_B03;SAD10319_P05_B04;SAD10319_P05_D01;SAD10319_P05_G02;SAD10319_P05_G03;SAD10319_P05_H06;SAD10319_P06_A07;SAD10319_P06_A10;SAD10319_P06_A11;SAD10319_P06_E09;SAD10319_P06_E10;SAD10319_P06_G11;SAD10319_P06_H11;LAD9953_P01_H01;LAD9954_P01_B02;LAD9955_P01_G02;LAD9956_P01_C03;LAD9959_P01_E04;LAD9960_P01_D05;LAD9963_P01_E06;LAD9964_P01_C07;LAD9966_P01_A08;SAD10318_P01_G01;SAD10318_P01_F02;SAD10318_P01_C03;SAD10318_P01_E03;SAD10318_P01_F03;SAD10318_P02_B04;SAD10318_P02_D04;SAD10318_P02_D06;SAD10318_P03_F07;SAD10318_P04_F11;SAD10318_P04_H12;SAD10319_P02_D04;SAD10319_P02_H04;SAD10319_P02_D05;SAD10319_P03_G07;SAD10319_P04_C10;SAD10319_P04_B11;SAD10319_P04_B12;SAD10320_P02_A06;SAD10319_P05_A03;SAD10319_P05_B05;SAD10319_P05_G04;SAD10319_P06_D12;SAD10318_P01_E01;SAD10318_P01_H01;SAD10318_P01_D02;SAD10318_P02_C04;SAD10318_P02_C05;SAD10318_P02_B06;SAD10318_P02_E06;SAD10318_P03_D09;SAD10318_P04_A12;SAD10319_P02_F05;SAD10319_P03_H08;SAD10320_P01_F01;SAD10320_P01_C02;SAD10320_P01_H03;SAD10320_P02_D05;SAD10320_P02_H05;SAD10320_P03_E07;SAD10320_P04_A11;SAD10319_P05_G01;SAD10318_P01_D01;SAD10318_P01_B02;SAD10318_P01_A03;SAD10318_P02_H04;SAD10318_P02_A05;SAD10318_P03_E07;SAD10318_P03_B08;SAD10318_P03_D08;SAD10318_P03_E08;SAD10318_P03_F08;SAD10318_P03_G08;SAD10318_P03_C09;SAD10318_P04_B11;SAD10319_P01_H01;SAD10319_P04_E12;SAD10318_P01_D01;SAD10318_P01_B02;SAD10318_P01_A03;SAD10318_P02_H04;SAD10318_P02_A05;SAD10318_P03_E07;SAD10318_P03_B08;SAD10318_P03_D08;SAD10318_P03_E08;SAD10318_P03_F08;SAD10318_P03_G08;SAD10318_P03_C09;SAD10318_P04_B11;SAD10319_P01_H01;SAD10319_P04_E12;SAD10318_P02_E04;SAD10318_P04_C11;SAD10318_P04_F12;SAD10319_P02_H05;SAD10320_P01_A03;SAD10320_P01_B03;SAD10320_P02_E05;ADI-26906;ADI-48584;ADI-57317;ADI-57319;ADI-57323;ADI-57328;ADI-48639;ADI-57300;ADI-57333;ADI-57336;ADI-57337;ADI-48587;ADI-57343;ADI-48648;ADI-48650;ADI-48589;ADI-48652;ADI-48654;ADI-48592;ADI-57401;ADI-57406;ADI-57274;ADI-57413;ADI-57414;ADI-5741; 5;ADI-57416;ADI-57417;ADI-57275;ADI-57427;ADI-57428;ADI-57437;ADI-57438;ADI-48594;ADI-57439;ADI-57440;ADI-57441 ;ADI-57442;ADI-57443;ADI-57444;ADI-57445;ADI-48666;ADI-48595;ADI-48597;ADI-48576;ADI-57277;ADI-57279;ADI-57280; ADI-57281;ADI-48601;ADI-48577;ADI-57284;ADI-48604;ADI-48606;ADI-57285;ADI-48608;ADI-48609;ADI-48610;ADI-48614;A DI-48615;ADI-48617;ADI-57295;ADI-48580;ADI-48622;ADI-57299;ADI-57300;ADI-48623;ADI-57303;ADI-48582; and ADI-57311.

[0202] In some embodiments, the disclosure provides antibodies comprising a CDRH3-binding domain comprising a consensus motif, the consensus motif comprising the sequence ARDAX1X2X3X4FYDX5, where Xi is T, H, or Y, X2 is G or H, X3 is H or R, X4 is V or Y, and X5 is V or H (SEQ ID NO: 593). In some embodiments, at least one of Xi, X2, X3, and X5 is H. At least the following six clones contain this consensus motif and are designated as Group 1 binders: ADI-48592, ADI-48595, ADI-48650, ADI-48652, ADI-48662, and ADI-48666.

[0203] In some embodiments, the disclosure provides an antibody comprising a CDRH3-binding domain comprising a consensus motif comprising the sequence AX1DX2X3X4X5X6X7YDX8, where Xi is R or H, X2 is H or A, X3 is H or Y, X4 is H, G or P, X5 is R or H, X6 is Y, I or V, X7 is F or H, and X8 is V or H (SEQ ID NO: 596). In some embodiments, at least one of Xi, X2, X3, X4, X5, X7, and X8 is H. At least the following 21 clones contain this consensus motif and are designated as group 2 binders: ADI-48588, ADI-48587, ADI-48577, ADI-48590, ADI-48581, ADI-48575, ADI-48593, ADI-48591, ADI-48647, ADI-48636, ADI-48586, ADI-48646, ADI-48638, ADI-48597, ADI-48601, ADI-48576, ADI-48643, ADI-48624, ADI-48632, ADI-48635, and ADI-48645.

[0204] In some embodiments, the present disclosure provides antibodies comprising a CDRH2-binding domain comprising a consensus motif, the consensus motif comprising the sequence WIDLENANTIYDAKFQG (SEQ ID NO: 9). The following 148 clones contain this consensus motif: LAD5224_P03_A01; SAD10318_P01_B01; SAD10318_P01_C01; SAD10318_P01_D01; SAD10318_P01_E01; SAD10318_P01_F01; SAD10318_P01_G01; SAD10318_P01_H01; SAD10318_P01_A02; SAD10318_P01_B02; SAD10318_P01_D02; SAD10318 _P01_F02;SAD10318_P01_G02;SAD10318_P01_A03;SAD10318_P01_C03;SAD10318_P01_D03;SAD10318_P01_E03;SAD10318_P01_F03;S AD10318_P01_G03;SAD10318_P01_H03;SAD10318_P02_B04;SAD10318_P02_C04;SAD10318_P02_D04;SAD10318_P02_E04;SAD10318_P0 2_G04;SAD10318_P02_H04;SAD10318_P02_A05;SAD10318_P02_B05;SAD10318_P02_C05;SAD10318_P02_D05;SAD10318_P02_F05;SAD1 0318_P02_G05;SAD10318_P02_H05;SAD10318_P02_B06;SAD10318_P02_D06;SAD10318_P02_E06;SAD10318_P02_G06;SAD10318_P03_B 07;SAD10318_P03_E07;SAD10318_P03_F07;SAD10318_P03_G07;SAD10318_P03_A08;SAD10318_P03_B08;SAD10318_P03_C08;SAD1031 8_P03_D08;SAD10318_P03_E08;SAD10318_P03_F08;SAD10318_P03_G08;SAD10318_P03_H08;SAD10318_P03_A09;SAD10318_P03_C09;SAD10318_P03_D09;SAD10318_P03_E09;SAD10318_P03_F09;SAD10318_P03_G09;SAD10318_P04_C10;SAD10318_P04_D10;SAD10318_P04_E10;SAD10318_P04_F10;SAD10318_P04_H10;SAD10318_P04_A11;SAD10318_P04_B11;SAD10318_P04_C11;SAD10318_P04_D11;SAD10318_P04_E11;SAD10318_P04_F11;SAD10318_P04_G11;SAD10318_P04_H11;SAD10318_P04_A12;SAD10318_P04_F12;SAD10318_P04_G12;SAD10318_P04_H12;SAD10320_P01_B01;SAD10320_P01_D01;SAD10320_P01_E01;SAD10320_P01_F01;SAD10320_P01_G01;SAD10320_P01_H01;SAD10320_P01_A02;SAD10320_P01_C02;SAD10320_P01_E02;SAD10320_P01_F02;SAD10320_P01_G02;SAD10320_P01_H02;SAD10320_P01_A03;SAD10320_P01_B03;SAD10320_P01_C03;SAD10320_P01_D03;SAD10320_P01_E03;SAD10320_P01_F03;SAD10320_P01_G03;SAD10320_P01_H03;SAD10320_P02_A04;SAD10320_P02_B04;SAD10320_P02_C04;SAD10320_P02_E04;SAD10320_P02_H04;SAD10320_P02_A05;SAD10320_P02_B05;SAD10320_P02_C05;SAD10320_P02_D05;SAD10320_P02_E05;SAD10320_P02_F05;SAD10320_P02_H05;SAD10320_P02_A06;SAD10320_P02_B06;SAD10320_P02_C06;SAD10320_P02_D06;SAD10320_P02_E06;SAD10320_P02_F06;SAD10320_P02_G06;SAD10320_P02_H06;SAD10320_P03_B07;SAD10320_P03_E 07;SAD10320_P03_H07;SAD10320_P03_C08;SAD10320_P03_D08;SAD10320_P03_F08;SAD10320_P0 3_H08;SAD10320_P03_A09;SAD10320_P03_C09;SAD10320_P03_D09;SAD10320_P03_F09;SAD10320 _P04_A10;SAD10320_P04_C10;SAD10320_P04_D10;SAD10320_P04_E10;SAD10320_P04_F10;SAD10 320_P04_G10;SAD10320_P04_A11;SAD10320_P04_D11;SAD10320_P04_E11;SAD10320_P04_F11;SA D10320_P04_G11;SAD10320_P04_H11;SAD10320_P04_A12;SAD10320_P04_D12;SAD10320_P04_E12 ;SAD10320_P04_F12;LAD9953_P01_H01;LAD9954_P01_B02;LAD9955_P01_G02;LAD9956_P01_C03;LAD9959_P01_E04;LAD9960_P01_D05;LAD9963_P01_E06;LAD9964_P01_C07;and LAD9966_P01_A08. Additionally, at least the following 16 clones contain this consensus motif and are designated as group 2 binders: ADI-48575, ADI-48576, ADI-48577, ADI-48581, ADI-48586, ADI-48587, ADI-48588, ADI-48590, ADI-48591, ADI-48593, ADI-48601, ADI-48646, ADI-48647, ADI-48597, ADI-48643, and ADI-48645;

[0205] In some embodiments, the disclosure provides antibodies comprising a CDRH2-binding domain comprising a consensus motif, the consensus motif comprising the sequence X1INPX2TGX3TX4YSQKFQG, where X1 is W or Y, X2 is A, S, D, G, N, L, V, H, or Q, X3 is A, T, or S, and X4 is K, V, T, D, Y, F, or A (SEQ ID NO: 10). In some embodiments, at least one of X1, X2, X3, and X4 is H. The following 24 clones contain this consensus motif: SAD10319_P01_E02; SAD10319_P01_H02; SAD10319_P01_B03; SAD10319_P02_A04. SAD10319_P02_B04;SAD10319_P02_C04;SAD10319_P02_F04;SAD10319_P02_H04;SAD10319_P02_A05; SAD10319_P02_C05;SAD10319_P02_C06;SAD10319_P02_E06;SAD10319_P02_F06;SAD10319_P02_G06;S AD10319_P03_D08;SAD10319_P03_F09;SAD10319_P04_G10;SAD10319_P04_C11;SAD10319_P05_A01;SAD10319_P05_A05;SAD10319_P05_G03;SAD10319_P06_A10;SAD10319_P06_C12; and SAD10319_P06_E09.

[0206] In some embodiments, the disclosure provides antibodies comprising a CDRH2-binding domain comprising a consensus motif comprising the sequence X1IX2AGTGX3TX4YSQKFQG, where X1 is W, Y, or F, X2 is T, N, or D, X3 is A, T, or L, and X4 is A, K, V, H, T, or N (SEQ ID NO: 11). In some embodiments, at least one of X1, X2, X3, and X4 is H. The following 23 clones contain this consensus motif: SAD10319_P01_E01; SAD10319_P01_G01; SAD10319_P01_D02; SAD10319_P01_D03; SAD10319_P02_E05; SAD10319_P02_A06; SAD10319_P03_C07; SAD10319_P03_G07; SAD10319_P03_B09; SAD10319_P03_E09; SAD10319_P04 _A10;SAD10319_P04_B10;SAD10319_P04_B11;SAD10319_P04_E12;SAD10319_P05_A02;SAD10319_P05_C05;SAD10319_P05_D01;SAD10319_P05_H06;SAD10319_P06_A07;SAD10319_P06_B11;SAD10319_P06_F07;SAD10319_P06_G09; and SAD10319_P06_H08.

[0207] In some embodiments, the disclosure provides antibodies comprising a CDRH2-binding domain comprising a consensus motif, the consensus motif comprising the sequence X1IDAGTGX2TX3YSQKFQG, where X1 is S or W, X2 is L, N, D, or F, and X3 is D, Y, or K (SEQ ID NO: 12). In some embodiments, at least one of X1, X2, and X3 is H. The following 17 clones contain this consensus motif: SAD10319_P01_C01; SAD10319_P01_D01; SAD10319_P01_H01; SAD10319_P01_F02; SAD10319_P02_D04; SAD10319_P02_D05; SAD10319_P02_F05; SAD10319_P02 _H06;SAD10319_P03_G08;SAD10319_P04_D11;SAD10319_P05_A03;SAD10319_P05_B05;SAD10319_P05_C01;SAD10319_P05_D03;SAD10319_P05_F01;SAD10319_P05_G01; and SAD10319_P06_H10.

[0208] In some embodiments, the disclosure provides antibodies comprising a CDRH2-binding domain comprising a consensus motif, the consensus motif comprising the sequence X1IX2AGTGATX3YSQKFQG, where X1 is G, D, or S, X2 is I or D, and X3 is K or D (SEQ ID NO: 13). In some embodiments, at least one of X1, X2, and X3 is H. The following seven clones contain this consensus motif: SAD10319_P02_G05; SAD10319_P05_B02; SAD10319_P05_C03; SAD10319_P05_D05; SAD10319_P06_B10; SAD10319_P06_C10; and SAD10319_P06_D12.

[0209] In some embodiments, the disclosure provides antibodies comprising a CDRH2-binding domain comprising a consensus motif, the consensus motif comprising the sequence WINPX1TGNTX2YSQKFQG, where X1 is D, T, L, S, or A, and X2 is D, V, L, or N (SEQ ID NO: 14). In some embodiments, at least one of X1 and X2 is H. The following six clones contain this consensus motif: SAD10319_P01_A01; SAD10319_P01_F01; SAD10319_P01_C02; SAD10319_P04_F12; SAD10319_P05_E04; and SAD10319_P06_A11.

[0210] In some embodiments, the disclosure provides antibodies comprising a CDRH2-binding domain comprising a consensus motif, the consensus motif comprising the sequence X1INAGTGX2TX3YSQKFQG, where X1 is Y or W, X2 is N, D, or A, and X3 is I or V (SEQ ID NO: 15). In some embodiments, at least one of X1, X2, and X3 is H. The following five clones contain this consensus motif: SAD10319_P01_F03; SAD10319_P02_H05; SAD10319_P02_D06; SAD10319_P03_E08; and SAD10319_P03_H08.

[0211] In some embodiments, the disclosure provides antibodies comprising a CDRH2-binding domain comprising a consensus motif, the consensus motif comprising the sequence X1INPX2TGX3TKYSQKFQG, where X1 is W or Y, X2 is D, I, or Y, and X3 is D, Y, or E (SEQ ID NO: 16). In some embodiments, at least one of X1, X2, and X3 is H. The following five clones contain this consensus motif: SAD10319_P03_H07; SAD10319_P04_E11; SAD10319_P04_F11; SAD10319_P04_B12; and SAD10319_P04_D12.

[0212] In some embodiments, the disclosure provides antibodies comprising a CDRH2-binding domain comprising a consensus motif, the consensus motif comprising the sequence SIX1AGTGX2TKYSQKFQG, where X1 is N or V, and X2 is A or I (SEQ ID NO: 17). In some embodiments, at least one of X1 and X2 is H. The following three clones contain this consensus motif: SAD10319_P02_E04; SAD10319_P04_C10; and SAD10319_P04_H12.

[0213] In some embodiments, the disclosure provides antibodies comprising a CDRH2-binding domain comprising a consensus motif, the consensus motif comprising the sequence SINAGTGX1TX2YSQKFQG, where X1 is F or N, and X2 is Y or D (SEQ ID NO: 18). In some embodiments, at least one of X1 and X2 is H. The following three clones contain this consensus motif: SAD10319_P02_B05; SAD10319_P02_B06; and SAD10319_P05_D02.

[0214] In some embodiments, the disclosure provides antibodies comprising a CDRH2-binding domain comprising a consensus motif, the consensus motif comprising the sequence X1IX2X3GTGX4TDYSQKFQG, where X1 is D or W, X2 is N or H, X3 is A or S, and X4 is A or N (SEQ ID NO: 19). In some embodiments, at least one of X1, X2, X3, and X4 is H. The following three clones contain this consensus motif: SAD10319_P05_B03; SAD10319_P05_B04; and SAD10319_P05_D04.

[0215] In some embodiments, the disclosure provides antibodies comprising a CDRH2-binding domain comprising a consensus motif, the consensus motif comprising the sequence WIDPX1TGATX2YSQKFQG, where X1 is N, H, or Y, and X2 is V or K (SEQ ID NO: 20). In some embodiments, at least one of X1 and X2 is H. The following three clones contain this consensus motif: SAD10319_P01_C03; SAD10319_P03_G09; and SAD10319_P06_F10.

[0216] In some embodiments, the disclosure provides antibodies comprising a CDRH2-binding domain comprising a consensus motif, the consensus motif comprising the sequence WIX1PX2TGNTKYSQKFQG, where X1 is D or N, and X2 is L, I, or V (SEQ ID NO: 21). In some embodiments, at least one of X1 and X2 is H. The following three clones contain this consensus motif: SAD10319_P01_A02; SAD10319_P04_C12; and SAD10319_P05_G02.

[0217] In some embodiments, the disclosure provides antibodies comprising a CDRH2-binding domain that comprises a consensus motif, the consensus motif comprising the sequence SINAGDANTKYSQKFQG (SEQ ID NO: 22). The following two clones contain this consensus motif: SAD10319_P04_G11 and SAD10319_P06_H07.

[0218] In some embodiments, the disclosure provides antibodies comprising a CDRH2-binding domain comprising a consensus motif, the consensus motif comprising the sequence X1IDPX2TGATX3YSQKFQG, where X1 is D or W, X2 is D or V, and X3 is E or D (SEQ ID NO: 23). In some embodiments, at least one of X1, X2, and X3 is H. The following two clones contain this consensus motif: SAD10319_P05_G04 and SAD10319_P06_E08.

[0219] In some embodiments, the disclosure provides antibodies comprising a CDRH2-binding domain that comprises a consensus motif, the consensus motif comprising the sequence WINAGDAATVYSQKFQG (SEQ ID NO: 24). The following two clones contain this consensus motif: SAD10319_P06_G11 and SAD10319_P06_H11.

[0220] In some embodiments, the disclosure provides antibodies comprising a CDRH2-binding domain comprising a consensus motif, the consensus motif comprising the sequence X1IX2X3X4X5X6X7TX8YSQKFQG, where X1 is W, S, Y, F, G, or D, X2 is N, T, D, V, or H, X3 is A, P, or S, X4 is G, A, S, N, D, L, V, H, Q, T, I, or Y, X5 is D or T, X6 is A or G, X7 is A, N, T, S, L, D, F, Y, or E, and X8 is V, K, T, D, Y, F, A, H, N, L, I, or E (SEQ ID NO: 59). In some embodiments, at least one of X1, X2, X3, X4, X5, X6, X7, and X8 is H. The following clones contain this consensus motif: SAD10319_P01_E02; SAD10319_P01_H02; SAD10319_P01_B03; SAD10319_P02_A04 SAD10319_P02_B04; SAD10319_P02_C04; SAD10319_P02_F04; SAD10319_P02_H04; SAD10319_P02_A05; SAD10319_P02_C05; SAD10319_P02_C06; SAD10319_P02_E06; SAD1 0319_P02_F06;SAD10319_P02_G06;SAD10319_P03_D08;SAD10319_P03_F09;SAD103 19_P04_G10;SAD10319_P04_C11;SAD10319_P05_A01;SAD10319_P05_A05;SAD10319 _P05_G03;SAD10319_P06_A10;SAD10319_P06_C12;SAD10319_P06_E09;SAD10319_P 01_E01;SAD10319_P01_G01;SAD10319_P01_D02;SAD10319_P01_D03;SAD10319_P02 _E05;SAD10319_P02_A06;SAD10319_P03_C07;SAD10319_P03_G07;SAD10319_P03_B 09;SAD10319_P03_E09;SAD10319_P04_A10;SAD10319_P04_B10;SAD10319_P04_B11;SAD10319_P04_E12;SAD10319_P05_A02;SAD10319_P05_C05;SAD10319_P05_D01;SAD10319_P05_H06;SAD10319_P06_A07;SAD10319_P06_B11;SAD10319_P06_F07;SAD10319_P06_G09;SAD10319_P06_H08;SAD10319_P01_C01;SAD10319_P01_D01;SAD10319_P01_H01;SAD10319_P01_F02;SAD10319_P02_D04;SAD10319_P02_D05;SAD10319_P02_F05;SAD10319_P02_H06;SAD10319_P03_G08;SAD10319_P04_D11;SAD10319_P05_A03;SAD10319_P05_B05;SAD10319_P05_C01;SAD10319_P05_D03;SAD10319_P05_F01;SAD10319_P05_G01;SAD10319_P06_H10;SAD10319_P02_G05;SAD10319_P05_B02;SAD10319_P05_C03;SAD10319_P05_D05;SAD10319_P06_B10;SAD10319_P06_C10;SAD10319_P06_D12;SAD10319_P01_A01;SAD10319_P01_F01;SAD10319_P01_C02;SAD10319_P04_F12;SAD10319_P05_E04;SAD10319_P06_A11;SAD10319_P01_F03;SAD10319_P02_H05;SAD10319_P02_D06;SAD10319_P03_E08;SAD10319_P03_H08;SAD10319_P03_H07;SAD10319_P04_E11;SAD10319_P04_F11;SAD10319_P04_B12;SAD10319_P04_D12;SAD10319_P02_E04;SAD10319_P04_C10;SAD10319_P04_H12;SAD10319_P02_B05;SAD10319_P02_B06;SAD10319_P05_D02;SAD10319_P05_B03;SAD10319_P05_B04;SAD10319_P05_D04;SAD10319_P01_C03;SAD10319_P03_G09;SAD10319_P06_F10;SAD10319_P01_A02;SAD10319_P04_C12;SAD10319_P05_G02;SAD10319_P04_G11;SAD10319_P06_H07;SAD10319_P05_G04;SAD10319_P06_E08;SAD10319_P06_G11; and SAD10319_P06_H11. ;

[0221] In some embodiments, the disclosure provides antibodies comprising a CDRH2-binding domain comprising a consensus motif, the consensus motif comprising the sequence WIDAGTGX1TX2YSQKFQG, where X1 is L, F, N, or A, and X2 is T or K (SEQ ID NO: 595). At least four clones contain this consensus motif and are designated as Group 2 binders: ADI-48636, ADI-48638, ADI-48624, and ADI-48635.

[0222] In some embodiments, the disclosure provides an antibody comprising a CDRH1-binding domain comprising a consensus motif, the consensus motif comprising the sequence X1NIKDYX2MH, where X1 is F or S and X2 is Y or H (SEQ ID NO: 44). In some embodiments, at least one of X1 and X2 is H. In some embodiments, the sequence is FNIKDYHMH (SEQ ID NO: 25), SNIKDYYMH (SEQ ID NO: 26), or SNIKDYHMH (SEQ ID NO: 27). The following 148 clones contain this consensus motif: LAD5224_P03_A01; SAD10318_P01_B01; SAD10318_P01_C01; SAD10318_P01_D01; SAD10318_P01_E01; SAD10318_P01_F01; SAD10318_P01_G01; SAD10318_P01_H01; SAD10318_P01_A02; SAD10318 _P01_B02;SAD10318_P01_D02;SAD10318_P01_F02;SAD10318_P01_G02;SAD10318_P01_A03;SAD10318_P01_C03;S AD10318_P01_D03;SAD10318_P01_E03;SAD10318_P01_F03;SAD10318_P01_G03;SAD10318_P01_H03;SAD10318_P0 2_B04;SAD10318_P02_C04;SAD10318_P02_D04;SAD10318_P02_E04;SAD10318_P02_G04;SAD10318_P02_H04;SAD1 0318_P02_A05;SAD10318_P02_B05;SAD10318_P02_C05;SAD10318_P02_D05;SAD10318_P02_F05;SAD10318_P02_G 05;SAD10318_P02_H05;SAD10318_P02_B06;SAD10318_P02_D06;SAD10318_P02_E06;SAD10318_P02_G06;SAD1031 8_P03_B07;SAD10318_P03_E07;SAD10318_P03_F07;SAD10318_P03_G07;SAD10318_P03_A08;SAD10318_P03_B08;SAD10318_P03_C08;SAD10318_P03_D08;SAD10318_P03_E08;SAD10318_P03_F08;SAD10318_P03_G08;SAD10318_P03_H08;SAD10318_P03_A09;SAD10318_P03_C09;SAD10318_P03_D09;SAD10318_P03_E09;SAD10318_P03_F09;SAD10318_P03_G09;SAD10318_P04_C10;SAD10318_P04_D10;SAD10318_P04_E10;SAD10318_P04_F10;SAD10318_P04_H10;SAD10318_P04_A11;SAD10318_P04_B11;SAD10318_P04_C11;SAD10318_P04_D11;SAD10318_P04_E11;SAD10318_P04_F11;SAD10318_P04_G11;SAD10318_P04_H11;SAD10318_P04_A12;SAD10318_P04_F12;SAD10318_P04_G12;SAD10318_P04_H12;SAD10320_P01_B01;SAD10320_P01_D01;SAD10320_P01_E01;SAD10320_P01_F01;SAD10320_P01_G01;SAD10320_P01_H01;SAD10320_P01_A02;SAD10320_P01_C02;SAD10320_P01_E02;SAD10320_P01_F02;SAD10320_P01_G02;SAD10320_P01_H02;SAD10320_P01_A03;SAD10320_P01_B03;SAD10320_P01_C03;SAD10320_P01_D03;SAD10320_P01_E03;SAD10320_P01_F03;SAD10320_P01_G03;SAD10320_P01_H03;SAD10320_P02_A04;SAD10320_P02_B04;SAD10320_P02_C04;SAD10320_P02_E04;SAD10320_P02_H04;SAD10320_P02_A05;SAD10320_P02_B05;SAD10320_P02_C05;SAD10320_P02_D05;SAD10320_P02_E05;SAD10320_P02_F05;SAD10320_P02_H05;SAD10320_P02_A06;SAD10320_P02_B06;SAD10320_P02_C 06;SAD10320_P02_D06;SAD10320_P02_E06;SAD10320_P02_F06;SAD10320_P02_G06;SAD10320_P02_H06;SAD10320_P0 3_B07;SAD10320_P03_E07;SAD10320_P03_H07;SAD10320_P03_C08;SAD10320_P03_D08;SAD10320_P03_F08;SAD10320 _P03_H08;SAD10320_P03_A09;SAD10320_P03_C09;SAD10320_P03_D09;SAD10320_P03_F09;SAD10320_P04_A10;SAD10 320_P04_C10;SAD10320_P04_D10;SAD10320_P04_E10;SAD10320_P04_F10;SAD10320_P04_G10;SAD10320_P04_A11;SA D10320_P04_D11;SAD10320_P04_E11;SAD10320_P04_F11;SAD10320_P04_G11;SAD10320_P04_H11;SAD10320_P04_A12 ;SAD10320_P04_D12;SAD10320_P04_E12;SAD10320_P04_F12;LAD9953_P01_H01;LAD9954_P01_B02;LAD9955_P01_G02;LAD9956_P01_C03;LAD9959_P01_E04;LAD9960_P01_D05;LAD9963_P01_E06;LAD9964_P01_C07;and LAD9966_P01_A08. ;

[0223] In some embodiments, the disclosure provides antibodies comprising a CDRH1-binding domain comprising a consensus motif, the consensus motif comprising the sequence YTFX1X2X3X4MH, where Xi is A, K, D, Q, E, N, T, L, Y, S, P, G, H, or V, X2 is T, S, or A, X3 is Y or I, and X4 is A, D, N, S, Y, T, I, V, L, E, P, R, or G (SEQ ID NO: 28). In some embodiments, at least one of Xi, X2, X3, and X4 is H. The following 61 clones contain this consensus motif: SAD10319_P01_A01; SAD10319_P01_D01; SAD10319_P01_E01; SAD10319_P01_F01; SAD10319_P01_F02; SAD10319_P01_B03; SAD10319_P01_D03; SAD10319_P01_F03; SAD10319_P02_A04; SAD 10319_P02_C04;SAD10319_P02_D04;SAD10319_P02_E04;SAD10319_P02_F04;SAD10319_P02_H04;SAD10319_P0 2_A05;SAD10319_P02_E05;SAD10319_P02_A06;SAD10319_P02_B06;SAD10319_P02_C06;SAD10319_P02_F06;SA D10319_P02_G06;SAD10319_P02_H06;SAD10319_P03_C07;SAD10319_P03_G07;SAD10319_P03_H07;SAD10319_P 03_D08;SAD10319_P03_G08;SAD10319_P03_H08;SAD10319_P03_E09;SAD10319_P03_F09;SAD10319_P03_G09;S AD10319_P04_B11;SAD10319_P04_C11;SAD10319_P04_D11;SAD10319_P04_G11;SAD10319_P04_B12;SAD10319_ P04_C12;SAD10319_P04_D12;SAD10319_P04_E12;SAD10319_P05_A01;SAD10319_P05_A02;SAD10319_P05_A05;SAD10319_P05_B02;SAD10319_P05_B03;SAD10319_P05_C03;SAD10319_P05_C05;SAD10319_P05_ D02;SAD10319_P05_G02;SAD10319_P05_G03;SAD10319_P05_G04;SAD10319_P06_A07;SAD10319_P 06_A11;SAD10319_P06_B11;SAD10319_P06_C12;SAD10319_P06_E09;SAD10319_P06_F07;SAD10319_P06_F10;SAD10319_P06_G09;SAD10319_P06_H07;SAD10319_P06_H08;and SAD10319_P06_H10. ;

[0224] In some embodiments, the disclosure provides antibodies comprising a CDRH1-binding domain comprising a consensus motif comprising the sequence YTFX1X2X3X4MH, where Xi is T, D, A, N, or V, X2 is D, E, G, or Q, X3 is Y or D, and X4 is D, A, E, N, S, Y, or V (SEQ ID NO: 29). In some embodiments, at least one of Xi, X2, X3, and X4 is H. The following 35 clones contain this consensus motif: SAD10319_P01_C01; SAD10319_P01_H01; SAD10319_P01_A02; SAD10319_P01_C02; SAD10319_P01_D02; SAD10319_P01_H02; SAD10319_P01_C03; SAD10319 _P02_B04;SAD10319_P02_B05;SAD10319_P02_C05;SAD10319_P02_D05;SAD10319_P02_D06;S AD10319_P02_E06;SAD10319_P03_E08;SAD10319_P03_B09;SAD10319_P04_C10;SAD10319_P04 _G10;SAD10319_P04_E11;SAD10319_P04_F11;SAD10319_P04_F12;SAD10319_P05_A03;SAD10 319_P05_B05;SAD10319_P05_C01;SAD10319_P05_D01;SAD10319_P05_D03;SAD10319_P05_D05 ;SAD10319_P05_E04;SAD10319_P05_F01;SAD10319_P05_H06;SAD10319_P06_A10;SAD10319_P06_B10;SAD10319_P06_C10;SAD10319_P06_E10;SAD10319_P06_G11;and SAD10319_P06_H11.

[0225] In some embodiments, the disclosure provides antibodies comprising a CDRH1-binding domain comprising a consensus motif, the consensus motif comprising the sequence YTFTSX1X2MH, where X1 is A, D, or T, and X2 is D, F, A, M, V, or Y (SEQ ID NO: 30). In some embodiments, at least one of X1 and X2 is H. The following seven clones contain this consensus motif: SAD10319_P01_G01; SAD10319_P01_E02; SAD10319_P04_A10; SAD10319_P04_B10; SAD10319_P04_H12; SAD10319_P05_B04; and SAD10319_P05_D04.

[0226] In some embodiments, the disclosure provides antibodies comprising a CDRH1-binding domain comprising a consensus motif, the consensus motif comprising the sequence YTFX1X2YX3MH, where X1 is N or T, X2 is Q or N, and X3 is S, T, or A (SEQ ID NO: 31). In some embodiments, at least one of X1, X2, and X3 is H. The following four clones contain this consensus motif: SAD10319_P02_F05; SAD10319_P02_G05; SAD10319_P02_H05; and SAD10319_P05_G01.

[0227] In some embodiments, the disclosure provides antibodies comprising a CDRH1-binding domain comprising a consensus motif, the consensus motif comprising the sequence YTFX1X2YVMH, where X1 is I or N, and X2 is K or R (SEQ ID NO: 32). In some embodiments, at least one of X1 and X2 is H. The following two clones contain this consensus motif: SAD10319_P06_D12 and SAD10319_P06_E08.

[0228] In some embodiments, the disclosure provides antibodies comprising a CDRH1-binding domain that comprises a consensus motif, the consensus motif comprising the sequence FNIKDYYMH (SEQ ID NO: 47). At least six clones contain this consensus motif and are designated as Group 1 binders: ADI-48592, ADI-48595, ADI-48650, ADI-48652, ADI-48662, and ADI-48666. Additionally, at least the following 16 clones contain this consensus motif and are designated as group 2 binders: ADI-48575, ADI-48576, ADI-48577, ADI-48581, ADI-48586, ADI-48587, ADI-48588, ADI-48590, ADI-48591, ADI-48593, ADI-48601, ADI-48646, ADI-48647, ADI-48597, ADI-48643, and ADI-48645.

[0229] In some embodiments, the disclosure provides antibodies comprising a CDRH1-binding domain comprising a consensus motif, the consensus motif comprising the sequence YTFX1X2YX3MH, where X1 is E, S, or T, X2 is S or D, and X3 is A or D (SEQ ID NO: 31). At least five clones contain this consensus motif and are designated as Group 2 binders: ADI-48636, ADI-48638, ADI-48624, ADI-48632, and ADI-48635.

[0230] In some embodiments, the disclosure provides antibodies comprising a CDRL3 binding domain comprising a consensus motif comprising the sequence X1X2SX3X4X5RX6, where X1 is H, K, or G, X2 is Q or H, X3 is Y or H, X4 is S, H, D, T, V, M, or L, X5 is R or H, and X6 is T or H (SEQ ID NO: 33). In some embodiments, at least one of X1, X2, X3, X4, and X5 is H. The following 156 clones contain the following consensus motif: LAD5224_P03_A01; SAD10318_P01_B01; SAD10318_P01_C01; SAD10318_P01_D01; SAD10318_P01_E01; SAD10318_P01_G01; SAD10318_P01_B02; SAD10318_P01_D02; SAD10318_P01_F02; SAD 10318_P01_G02;SAD10318_P01_A03;SAD10318_P01_C03;SAD10318_P01_D03;SAD10318_P01_E03;SAD10318_P0 1_F03;SAD10318_P02_B04;SAD10318_P02_C04;SAD10318_P02_D04;SAD10318_P02_E04;SAD10318_P02_H04;SA D10318_P02_A05;SAD10318_P02_B05;SAD10318_P02_C05;SAD10318_P02_F05;SAD10318_P02_G05;SAD10318_P 02_H05;SAD10318_P02_D06;SAD10318_P02_E06;SAD10318_P02_G06;SAD10318_P03_E07;SAD10318_P03_F07;S AD10318_P03_A08;SAD10318_P03_B08;SAD10318_P03_C08;SAD10318_P03_D08;SAD10318_P03_E08;SAD10318_ P03_F08;SAD10318_P03_G08;SAD10318_P03_H08;SAD10318_P03_D09;SAD10318_P03_G09;SAD10318_P04_D10;SAD10318_P04_F10;SAD10318_P04_H10;SAD10318_P04_B11;SAD10318_P04_C11;SAD10318_P04_D11;SAD10318_P04_E11;SAD10318_P04_F11;SAD10318_P04_G11;SAD10318_P04_H11;SAD10318_P04_F12;SAD10318_P04_G12;SAD10318_P04_H12;SAD10319_P01_A01;SAD10319_P01_C01;SAD10319_P01_D01;SAD10319_P01_E01;SAD10319_P01_F01;SAD10319_P01_G01;SAD10319_P01_H01;SAD10319_P01_A02;SAD10319_P01_C02;SAD10319_P01_D02;SAD10319_P01_E02;SAD10319_P01_F02;SAD10319_P01_H02;SAD10319_P01_B03;SAD10319_P01_C03;SAD10319_P01_D03;SAD10319_P01_F03;SAD10319_P02_A04;SAD10319_P02_B04;SAD10319_P02_C04;SAD10319_P02_D04;SAD10319_P02_E04;SAD10319_P02_F04;SAD10319_P02_H04;SAD10319_P02_A05;SAD10319_P02_B05 SAD10319_P02_C05;SAD10319_P02_D05;SAD10319_P02_E05;SAD10319_P02_F05;SAD10319_P02_G05;SAD10319_P02_H05;SAD10319_P02_A06;SAD10319_P02_B06;SAD10319_P02_C06;SAD10319_P02_D06;SAD10319_P02_E06;SAD10319_P02_F06;SAD10319_P02_G06;SAD10319_P02_H06;SAD10319_P03_C07;SAD10319_P03_G07;SAD10319_P03_H07;SAD10319_P03_D08;SAD10319_P03_E08;SAD10319_P03_G08;SAD10319_P03_H08;SAD10319_P03_B09;SAD10319_P03_E09;SAD10319_P03_F09;SAD10319_P03_G09;SAD10319_P04_A10;SAD10319_P04_B10 ;SAD10319_P04_C10;SAD10319_P04_G10;SAD10319_P04_B11;SAD10319_P04_C11;SAD10319_P04_D11;SAD10319_P04_E11;SAD10319_P04_F1 1;SAD10319_P04_G11;SAD10319_P04_B12;SAD10319_P04_C12;SAD10319_P04_D12;SAD10319_P04_E12;SAD10319_P04_F12;SAD10319_P04_H 12;SAD10320_P01_D01;SAD10320_P01_F01;SAD10320_P01_C02;SAD10320_P01_E02;SAD10320_P01_B03;SAD10320_P01_H03;SAD10320_P02_C 04;SAD10320_P02_E04;SAD10320_P02_H04;SAD10320_P02_B05;SAD10320_P02_D05;SAD10320_P02_H05;SAD10320_P02_A06;SAD10320_P02_ E06;SAD10320_P02_F06;SAD10320_P02_G06;SAD10320_P03_B07;SAD10320_P03_H07;SAD10320_P03_F08;SAD10320_P04_A10;SAD10320_P04 _E10;SAD10320_P04_G10;SAD10320_P04_A11;SAD10320_P04_F11;SAD10320_P04_D12;SAD10320_P04_F12;LAD9953_P01_H01;LAD9954_P01_B02;LAD9955_P01_G02;LAD9956_P01_C03;LAD9959_P01_E04;LAD9960_P01_D05;LAD9963_P01_E06;LAD9964_P01_C07;and LAD9966_P01_A08. ;

[0231] In some embodiments, the disclosure provides antibodies comprising a CDRL3 binding domain comprising a consensus motif comprising the sequence KQSYX1X2RT, where X1 is H, V, K, W, R, L, G, Y, or Q, and X2 is H, L, E, W, G, M, P, T, Q, or V (SEQ ID NO: 34). In some embodiments, at least one of X1 and X2 is H. The following 45 clones contain this consensus motif: SAD10318_P01_F01; SAD10318_P01_H01; SAD10318_P01_A02; SAD10318_P01_G03; SAD10318_P01_H03; SAD10318_P02_G04; SAD10318_P02_D05; SAD10318_P02_B06; SAD10318_P03_G07; SAD10318_P03_A09; SAD10318_P03_C09;SAD10318_P03_E09;SAD10318_P04_A12;SAD10320_P01_B01;SAD10320_P01_E01;SAD10320_P01_G 01;SAD10320_P01_A02;SAD10320_P01_F02;SAD10320_P01_G02;SAD10320_P01_C03;SAD10320_P01_D03;SAD10320_P01 _E03;SAD10320_P01_F03;SAD10320_P01_G03;SAD10320_P02_B04;SAD10320_P02_A05;SAD10320_P02_C05;SAD10320_ P02_F05;SAD10320_P02_B06;SAD10320_P02_D06;SAD10320_P02_H06;SAD10320_P03_E07;SAD10320_P03_C08;SAD1032 0_P03_D08;SAD10320_P03_H08;SAD10320_P03_C09;SAD10320_P04_C10;SAD10320_P04_D10;SAD10320_P04_F10;SAD10320_P04_D11;SAD10320_P04_E11;SAD10320_P04_G11;SAD10320_P04_H11;SAD10320_P04_A12; and SAD10320_P04_E12.

[0232] In some embodiments, the disclosure provides antibodies comprising a CDRL3 binding domain comprising a consensus motif comprising the sequence X1QSX2HX3RT, where X1 is K or H, X2 is H, Y, M, S, L, E, G, or W, and X3 is R or K (SEQ ID NO: 35). In some embodiments, at least one of X1, X2, and X3 is H. The following 14 clones contain this consensus motif: SAD10318_P03_B07; SAD10318_P03_F09; SAD10318_P04_C10; SAD10318_P04_E10; SAD10318_P04_A11; SAD10320_P01_H01; SAD10320_P01_H02; SAD10320_P01_A03; SAD10320_P02_A04; SAD10320_P02_E05; SAD10320_P02_C06; SAD10320_P03_A09; SAD10320_P03_D09; and SAD10320_P03_F09.

[0233] In some embodiments, the disclosure provides antibodies comprising a CDRL3-binding domain comprising a consensus motif, the consensus motif comprising the sequence KQSX1X2X3RT, where X1 is Y or H, X2 is T, S, V, or K, and X3 is R or H (SEQ ID NO: 36). In some embodiments, at least one of X1, X2, and X3 is H. The following 11 clones contain this consensus motif: ADI-48576; ADI-48577; ADI-48587; ADI-48592; ADI-48595; ADI-48635; ADI-48650; ADI-48652; ADI-48666; ADI-48645; and ADI-48643.

[0234] In some embodiments, the disclosure provides antibodies comprising a CDRL3-binding domain comprising a consensus motif, the consensus motif comprising the sequence KQSX1X2X3RT, where X1 is H or Y, X2 is T, S, or Q, and X3 is R or H (SEQ ID NO: 36). In some embodiments, at least one of X1 and X3 is H. At least six clones contain this consensus motif and are designated as Group 1 binders: ADI-48592, ADI-48595, ADI-48650, ADI-48652, ADI-48662, and ADI-48666.

[0235] In some embodiments, the disclosure provides an antibody comprising a CDRL3 binding domain comprising a consensus motif comprising the sequence X1QSX2X3X4RT, where X1 is K or H, X2 is Y or H, X3 is S, H, L, V, or K, and X4 is H, R, or E (SEQ ID NO: 598). In some embodiments, at least one of X1, X2, X3, and X4 is H. At least the following 21 clones contain this consensus motif and are designated as group 2 binders: ADI-48588, ADI-48587, ADI-48577, ADI-48590, ADI-48581, ADI-48575, ADI-48593, ADI-48591, ADI-48647, ADI-48636, ADI-48586, ADI-48646, ADI-48638, ADI-48597, ADI-48601, ADI-48576, ADI-48643, ADI-48624, ADI-48632, ADI-48635, and ADI-48645.

[0236] In some embodiments, the present disclosure provides antibodies comprising a CDRL2-binding domain comprising a consensus motif, the consensus motif comprising the sequence WASTRES (SEQ ID NO: 37). The following 215 clones contain this consensus motif: LAD5224_P03_A01; SAD10318_P01_B01; SAD10318_P01_C01; SAD10318_P01_D01; SAD10318_P01_E01; SAD10318_P01_F01; SAD10318_P01_G01; SAD10318_P01_H01; SAD10318_P01_A02; SAD10318_P01_B02; SAD10318_P01_D02; SAD10318 _P01_F02;SAD10318_P01_G02;SAD10318_P01_A03;SAD10318_P01_C03;SAD10318_P01_D03;SAD10318_P01_E03;SAD10318_P01_F03;S AD10318_P01_G03;SAD10318_P01_H03;SAD10318_P02_B04;SAD10318_P02_C04;SAD10318_P02_D04;SAD10318_P02_E04;SAD10318_P0 2_G04;SAD10318_P02_H04;SAD10318_P02_A05;SAD10318_P02_B05;SAD10318_P02_C05;SAD10318_P02_D05;SAD10318_P02_F05;SAD1 0318_P02_G05;SAD10318_P02_H05;SAD10318_P02_B06;SAD10318_P02_D06;SAD10318_P02_E06;SAD10318_P02_G06;SAD10318_P03_B 07;SAD10318_P03_E07;SAD10318_P03_F07;SAD10318_P03_G07;SAD10318_P03_A08;SAD10318_P03_B08;SAD10318_P03_C08;SAD1031 8_P03_D08;SAD10318_P03_E08;SAD10318_P03_F08;SAD10318_P03_G08;SAD10318_P03_H08;SAD10318_P03_A09;SAD10318_P03_C09;SAD10318_P03_D09;SAD10318_P03_E09;SAD10318_P03_F09;SAD10318_P03_G09;SAD10318_P04_C10;SAD10318_P04_D10;SAD10318_P04_E10;SAD10318_P04_F10;SAD10318_P04_H10;SAD10318_P04_A11;SAD10318_P04_B11;SAD10318_P04_C11;SAD10318_P04_D11;SAD10318_P04_E11;SAD10318_P04_F11;SAD10318_P04_G11;SAD10318_P04_H11;SAD10318_P04_A12;SAD10318_P04_F12;SAD10318_P04_G12;SAD10318_P04_H12;SAD10319_P01_A01;SAD10319_P01_C01;SAD10319_P01_D01;SAD10319_P01_E01;SAD10319_P01_F01;SAD10319_P01_G01;SAD10319_P01_H01;SAD10319_P01_A02;SAD10319_P01_C02;SAD10319_P01_D02;SAD10319_P01_E02;SAD10319_P01_F02;SAD10319_P01_H02;SAD10319_P01_B03;SAD10319_P01_C03;SAD10319_P01_D03;SAD10319_P01_F03;SAD10319_P02_A04;SAD10319_P02_B04;SAD10319_P02_C04;SAD10319_P02_D04;SAD10319_P02_E04;SAD10319_P02_F04;SAD10319_P02_H04;SAD10319_P02_A05;SAD10319_P02_B05;SAD10319_P02_C05;SAD10319_P02_D05;SAD10319_P02_E05;SAD10319_P02_F05;SAD10319_P02_G05;SAD10319_P02_H05;SAD10319_P02_A06;SAD10319_P02_B06;SAD10319_P02_C06;SAD10319_P02_D06;SAD10319_P02_E06;SAD10319_P02_F06;SAD10319_P02_G06;SAD10319_P02_H06;SAD10319_P03_C07;SAD10319_P03_G07;SAD10319_P03_H07;SAD10319_P03_D08;SAD10319_P03_E08;SAD10319_P03_G08;SAD10319_P03_H08;SAD10319_P03_B09;SAD10319_P03_E09;SAD10319_P03_F09;SAD10319_P03_G09;SAD10319_P04_A10;SAD10319_P04_B10;SAD10319_P04_C10;SAD10319_P04_G10;SAD10319_P04_B11;SAD10319_P04_C11;SAD10319_P04_D11;SAD10319_P04_E11;SAD10319_P04_F11;SAD10319_P04_G11;SAD10319_P04_B12;SAD10319_P04_C12;SAD10319_P04_D12;SAD10319_P04_E12;SAD10319_P04_F12;SAD10319_P04_H12;SAD10320_P01_B01;SAD10320_P01_D01;SAD10320_P01_E01;SAD10320_P01_F01;SAD10320_P01_G01;SAD10320_P01_H01;SAD10320_P01_A02;SAD10320_P01_C02;SAD10320_P01_E02;SAD10320_P01_F02;SAD10320_P01_G02;SAD10320_P01_H02;SAD10320_P01_A03;SAD10320_P01_B03;SAD10320_P01_C03;SAD10320_P01_D03;SAD10320_P01_E03;SAD10320_P01_F03;SAD10320_P01_G03;SAD10320_P01_H03;SAD10320_P02_A04;SAD10320_P02_B04;SAD10320_P02_C04;SAD10320_P02_E04;SAD10320_P02_H04;SAD10320_P02_A05;SAD10320_P02_B05;SAD10320_P02_C05;SAD10320_P02_D05;SAD10320_P02_E05;SAD10320_P02_F05;SAD10320_P02_H05;SAD10320_P02_A06;SAD10320_P02_B06;SAD10320_P02_C06;SAD10320_P02_D06;SAD10320_P02_E06;SAD10320_P02_F06;SAD10320_P02_G06;SAD10320_P02_H06;SAD10320_P03_B07;SAD10320_P03_E07;SAD10320_P03_H07;SAD10320_P03_C08;SAD10320_P03_D08;SAD10320_P03_F08;SAD10320_P03_H08;SAD10320_P03_A09;SAD10320_P03_C09;SAD10320_P03_D09;SAD10320_P03_F09;SAD10320_P04_A10;SAD10320_P04_C10;SAD10320_P04_D10;SAD10320_P04_E10;SAD10320_P04_F10;SAD10320_P04_G10;SAD10320_P04_A11;SAD10320_P04_D11;SAD10320_P04_E11;SAD10320_P04_F11;SAD10320_P04_G11;SAD10320_P04_H11;SAD10320_P04_A12;SAD10320_P04_D12;SAD10320_P04_E12;SAD10320_P04_F12;LAD9953_P01_H01;LAD9954_P01_B02;LAD9955_P01_G02;LAD9956_P01_C03;LAD9959_P01_E04;LAD9960_P01_D05;LAD9963_P01_E06;LAD9964_P01_C07;and LAD9966_P01_A08. Additionally, at least the following six clones contain this consensus motif and are designated as Group 1 binders: ADI-48592, ADI-48595, ADI-48650, ADI-48652, ADI-48662, and ADI-48666. At least the following 21 clones contain this consensus motif and are designated as group 2 binders: ADI-48588, ADI-48587, ADI-48577, ADI-48590, ADI-48581, ADI-48575, ADI-48593, ADI-48591, ADI-48647, ADI-48636, ADI-48586, ADI-48646, ADI-48638, ADI-48597, ADI-48601, ADI-48576, ADI-48643, ADI-48624, ADI-48632, ADI-48635, and ADI-48645;

[0237] In some embodiments, the disclosure provides an antibody comprising a CDRL1-binding domain comprising a consensus motif comprising the sequence KSSQSLLX1X2X3X4GX5NX6LA, where X1 is N or H, X2 is A, R or T, X3 is R or H, X4 is T, P or E, X5 is H or K, and X6 is H or Y (SEQ ID NO: 38). In some embodiments, at least one of X1, X2, X3, X4, X5, and X6 is H. The following 203 clones contain this consensus motif: LAD5224_P03_A01; SAD10318_P01_B01; SAD10318_P01_D01; SAD10318_P01_E01; SAD10318_P01_F01; SAD10318_P01_G01; SAD10318_P01_H01; SAD10318_P01_B02; SAD10318_P01_D02; SAD 10318_P01_F02;SAD10318_P01_G02;SAD10318_P01_A03;SAD10318_P01_C03;SAD10318_P01_D03;SAD10318_P0 1_E03;SAD10318_P01_F03;SAD10318_P01_G03;SAD10318_P01_H03;SAD10318_P02_B04;SAD10318_P02_C04;SA D10318_P02_D04;SAD10318_P02_E04;SAD10318_P02_G04;SAD10318_P02_H04;SAD10318_P02_A05;SAD10318_P 02_B05;SAD10318_P02_C05;SAD10318_P02_D05;SAD10318_P02_F05;SAD10318_P02_H05;SAD10318_P02_B06;S AD10318_P02_D06;SAD10318_P02_G06;SAD10318_P03_B07;SAD10318_P03_E07;SAD10318_P03_F07;SAD10318_ P03_G07;SAD10318_P03_A08;SAD10318_P03_B08;SAD10318_P03_C08;SAD10318_P03_D08;SAD10318_P03_E08;SAD10318_P03_F08;SAD10318_P03_G08;SAD10318_P03_H08;SAD10318_P03_A09;SAD10318_P03_C09;SAD10318_P03_D09;SAD10318_P03_E09;SAD10318_P03_F09;SAD10318_P03_G09;SAD10318_P04_C10;SAD10318_P04_D10;SAD10318_P04_E10;SAD10318_P04_H10;SAD10318_P04_A11;SAD10318_P04_B11;SAD10318_P04_C11;SAD10318_P04_D11;SAD10318_P04_E11;SAD10318_P04_F11;SAD10318_P04_G11;SAD10318_P04_H11;SAD10318_P04_A12;SAD10318_P04_F12;SAD10318_P04_G12;SAD10318_P04_H12;SAD10319_P01_A01;SAD10319_P01_C01;SAD10319_P01_D01;SAD10319_P01_E01;SAD10319_P01_F01;SAD10319_P01_G01;SAD10319_P01_H01;SAD10319_P01_A02;SAD10319_P01_C02;SAD10319_P01_D02;SAD10319_P01_E02;SAD10319_P01_F02;SAD10319_P01_H02;SAD10319_P01_B03;SAD10319_P01_C03;SAD10319_P01_D03;SAD10319_P01_F03;SAD10319_P02_A04;SAD10319_P02_B04;SAD10319_P02_C04;SAD10319_P02_D04;SAD10319_P02_E04;SAD10319_P02_F04;SAD10319_P02_H04;SAD10319_P02_A05;SAD10319_P02_B05;SAD10319_P02_C05;SAD10319_P02_D05;SAD10319_P02_E05;SAD10319_P02_F05;SAD10319_P02_G05;SAD10319_P02_H05;SAD10319_P02_A06;SAD10319_P02_B06;SAD10319_P02_C06;SAD10319_P02_D06;SAD10319_P02_E06;SAD10319_P02_F06;SAD10319_P02_G06;SAD10319_P02_H06;SAD10319_P03_C07;SAD10319_P03_G07;SAD10319_P03_H07;SAD10319_P03_D08;SAD10319_P03_E08;SAD10319_P03_G08;SAD10319_P03_H08;SAD10319_P03_B09;SAD10319_P03_E09;SAD10319_P03_F09;SAD10319_P03_G09;SAD10319_P04_A10;SAD10319_P04_B10;SAD10319_P04_C10;SAD10319_P04_G10;SAD10319_P04_B11;SAD10319_P04_C11;SAD10319_P04_D11;SAD10319_P04_E11;SAD10319_P04_F11;SAD10319_P04_G11;SAD10319_P04_B12;SAD10319_P04_C12;SAD10319_P04_D12;SAD10319_P04_E12;SAD10319_P04_F12;SAD10319_P04_H12;SAD10320_P01_B01;SAD10320_P01_D01;SAD10320_P01_E01;SAD10320_P01_F01;SAD10320_P01_G01;SAD10320_P01_H01;SAD10320_P01_A02;SAD10320_P01_C02;SAD10320_P01_E02;SAD10320_P01_F02;SAD10320_P01_G02;SAD10320_P01_H02;SAD10320_P01_A03;SAD10320_P01_B03;SAD10320_P01_C03;SAD10320_P01_D03;SAD10320_P01_E03;SAD10320_P01_F03;SAD10320_P01_G03;SAD10320_P01_H03;SAD10320_P02_A04;SAD10320_P02_B04;SAD10320_P02_C04;SAD10320_P02_E04;SAD10320_P02_H04;SAD10320_P02_A05;SAD10320_P02_B05;SAD10320_P02_C05;SAD10320_P02_D05;SAD10320_P 02_E05;SAD10320_P02_F05;SAD10320_P02_H05;SAD10320_P02_A06;SAD10320_P02_B06;SAD10320_P02_C06;SAD1 0320_P02_D06;SAD10320_P02_E06;SAD10320_P02_F06;SAD10320_P02_H06;SAD10320_P03_B07;SAD10320_P03_E 07;SAD10320_P03_H07;SAD10320_P03_C08;SAD10320_P03_D08;SAD10320_P03_F08;SAD10320_P03_A09;SAD10320 _P03_C09;SAD10320_P03_D09;SAD10320_P03_F09;SAD10320_P04_A10;SAD10320_P04_C10;SAD10320_P04_D10;S AD10320_P04_E10;SAD10320_P04_F10;SAD10320_P04_G10;SAD10320_P04_A11;SAD10320_P04_D11;SAD10320_P04 _E11;SAD10320_P04_F11;SAD10320_P04_G11;SAD10320_P04_H11;SAD10320_P04_A12;SAD10320_P04_D12;SAD10320_P04_E12;SAD10320_P04_F12;LAD9954_P01_B02;LAD9955_P01_G02;LAD9963_P01_E06;and LAD9966_P01_A08. ;

[0238] In some embodiments, the disclosure provides an antibody comprising a CDRL1-binding domain comprising a consensus motif comprising the sequence KSSQSLLX1AX2THX3NX4LA, where X1 is N or H, X2 is R or H, X3 is K or H, and X4 is Y or H (SEQ ID NO: 39). In some embodiments, at least one of X1, X2, X3, and X4 is H. The following 10 clones contain this consensus motif: SAD10318_P01_C01; SAD10318_P01_A02; SAD10318_P02_G05; SAD10318_P02_E06; SAD10318_P04_F10; LAD9953_P01_H01; LAD9956_P01_C03; LAD9959_P01_E04; LAD9960_P01_D05; and LAD9964_P01_C07.

[0239] In some embodiments, the disclosure provides an antibody comprising a CDRL1-binding domain comprising a consensus motif, wherein the consensus motif comprises the sequence KSSQSLLNASTAKNYLA (SEQ ID NO: 40) or KSSQSLLNARTRTNYLA (SEQ ID NO: 41).

[0240] In some embodiments, the disclosure provides antibodies comprising a CDRL1-binding domain comprising a consensus motif, the consensus motif comprising the sequence KSSQSLLNX1X2X3GX4NX5LA, where X1 is S or A, X2 is R or H, X3 is E or T, X4 is H or K, and X5 is H or Y (SEQ ID NO: 42). In some embodiments, at least one of X1, X2, X3, X4, and X5 is H. The following 11 clones contain this consensus motif: ADI-48576; ADI-48577; ADI-48587; ADI-48592; ADI-48595; ADI-48635; ADI-48645; ADI-48650; ADI-48652; ADI-48643; and ADI-48666.

[0241] In some embodiments, the disclosure provides antibodies comprising a CDRL1-binding domain comprising a consensus motif, the consensus motif comprising the sequence KSSQSLLNX1X2TGX3NYLA, where X1 is A or S, X2 is R or H, and X3 is H or K (SEQ ID NO: 594). In some embodiments, at least one of X2 and X3 is H. At least six clones contain this consensus motif and are designated as Group 1 binders: ADI-48592, ADI-48595, ADI-48650, ADI-48652, ADI-48662, and ADI-48666.

[0242] In some embodiments, the disclosure provides an antibody comprising a CDRL1-binding domain comprising a consensus motif comprising the sequence KSSQSLLX1AX2X3X4X5NX6LA, where X1 is N or H, X2 is R or H, X3 is T or E, X4 is G or H, X5 is H or K, and X6 is H or Y (SEQ ID NO: 597). In some embodiments, at least one of X1, X2, X4, X5, and X6 is H. At least the following 21 clones contain this consensus motif and are designated as group 2 binders: ADI-48588, ADI-48587, ADI-48577, ADI-48590, ADI-48581, ADI-48575, ADI-48593, ADI-48591, ADI-48647, ADI-48636, ADI-48586, ADI-48646, ADI-48638, ADI-48597, ADI-48601, ADI-48576, ADI-48643, ADI-48624, ADI-48632, ADI-48635, and ADI-48645.

[0243] In some embodiments, the disclosure provides a CDRH3 binding domain comprising the consensus motif AX1DX2YX3HX4FYDV, where X1 is R or H, X2 is A or H, X3 is G, H or P, and X4 is Y, H, D, V, E, S, N, L, M, I, G, A, Q, or T (SEQ ID NO: 1); a CDRH2 binding domain comprising the consensus motif WIDLENANTIYDAKFQG, where X1 is S, D, A, N, L, or Q, and X2 is V, T, D, Y, or K (SEQ ID NO: 45); or an X1IDAGTGATX2YSQKFQG, where X1 is W, S, or D and X2 is a CDRH1 binding domain comprising a consensus motif, wherein X1 is S, H, V, K, W, L, G, T, R, or Q, and X2 is H, R, L, K, E, W, G, M, T, or V (SEQ ID NO: 48); a CDRL2 binding domain comprising a consensus motif, wherein X1 is S, H, V, K, W, L, G, T, R, or Q, and X2 is H, R, L, K, E, W, G, M, T, or V (SEQ ID NO: 37); and a CDRL1 binding domain comprising a consensus motif, wherein the consensus motif comprises the sequence KSSQSLLNARTGKNYLA (SEQ ID NO: 49).

[0244] In some embodiments, the disclosure provides a CDRH3-binding domain comprising a consensus motif comprising the sequence ARDX1YGX2X3X4YDX5, where X1 is A or H, X2 is R or H, X3 is H or Y, X4 is F or H, and X5 is H or V (SEQ ID NO: 2); a CDRH2-binding domain comprising a consensus motif comprising the sequence WIDLENANTIYDAKFQG (SEQ ID NO: 9) or the sequence WIX1AGTGATX2YSQKGQG, where X1 is T, N, or D, and X2 is V or K (SEQ ID NO: 50); a CDRH1-binding domain comprising a consensus motif comprising the sequence FNIKDYYMH (SEQ ID NO: 47) or the sequence YTFX1X2YX3MH, wherein X1 is T or A, X2 is E, D, A, S, G or Q, and X3 is D, A, V or E (SEQ ID NO: 51); a CDRL3 binding domain comprising a consensus motif, the consensus motif comprising the sequence KQSX1SRRT, where X1 is H or Y (SEQ ID NO: 52); a CDRH2 binding domain comprising a consensus motif, the consensus motif comprising the sequence WASTRES (SEQ ID NO: 37); and a CDRL1 binding domain comprising a consensus motif, the consensus motif comprising the sequence KSSQSLLX1AX2TX3X4NX5LA, where X1 is N or H, X2 is R or H, X3 is G or H, X4 is K or H, and X5 is H or Y (SEQ ID NO: 53).

[0245] In some embodiments, the disclosure provides a CDRH3 binding domain comprising a consensus motif, the consensus motif comprising the sequence ARDAHX1X2YX3X4DX5, where X1 is G, E, or R, X2 is R or H, X3 is F or H, X4 is Y or H, and X5 is V or H (SEQ ID NO: 3); a CDRH2 binding domain comprising a consensus motif, the consensus motif comprising the sequence WIDLENANTIYDAKFQG (SEQ ID NO: 9); a CDRH1 binding domain comprising a consensus motif, the consensus motif comprising the sequence FNIKDYYMH (SEQ ID NO: 9); a CDRH2 binding domain comprising a consensus motif comprising the sequence KSSQSLLNAX1TGX2NX3LA, where X1 is H or R, X2 is H or K, and X3 is H or Y (SEQ ID NO: 55); a CDRL3 binding domain comprising a consensus motif comprising the sequence KQSYSRRT (SEQ ID NO: 54); a CDRH2 binding domain comprising a consensus motif comprising the sequence WASTRES (SEQ ID NO: 37); and a CDRL1 binding domain comprising a consensus motif comprising the sequence KSSQSLLNAX1TGX2NX3LA, where X1 is H or R, X2 is H or K, and X3 is H or Y (SEQ ID NO: 55).

[0246] In some embodiments, the disclosure provides a CDRH3 binding domain comprising a consensus motif, the consensus motif comprising the sequence ARDAX1X2X3X4FYDX5, where Xi is T, H, or Y, X2 is G or H, X3 is H or R, X4 is V or Y, X5 is V or H, and optionally, at least one of Xi, X2, X3, and X5 is H (SEQ ID NO: 593); a CDRH2 binding domain comprising a consensus motif, the consensus motif comprising the sequence WIDLENANTIYDAKFQG (SEQ ID NO: 9); a CDRH1 binding domain comprising a consensus motif, the consensus motif comprising the sequence FNIKDYYMH (SEQ ID NO: 47); a CDRL3 binding domain comprising a consensus motif. wherein the consensus motif comprises the sequence KQSX1X2X3RT, where X1 is H or Y, X2 is T, S, or Q, and X3 is R or H, and optionally, at least one of X1 and X3 is H (SEQ ID NO: 36); a CDRH2 binding domain comprising the consensus motif, where the consensus motif comprises the sequence WASTRES (SEQ ID NO: 37); and / or a CDRL1 binding domain comprising the consensus motif, where the consensus motif comprises the sequence KSSQSLLNX1X2TGX3NYLA, where X1 is A or S, X2 is R or H, and X3 is H or K, and optionally, at least one of X2 and X3 is H (SEQ ID NO: 594). In some embodiments, the antibody or antigen-binding fragment is designated as a Group 1 binder, comprising a CD3-binding domain selected from ADI-48592, ADI-48595, ADI-48650, ADI-48652, ADI-48662, and ADI-48666.

[0247] In some embodiments, the disclosure provides a CDRH3 binding domain comprising a consensus motif, the consensus motif comprising the sequence AX1DX2X3X4X5X6X7YDX8, where X1 is R or H, X2 is H or A, X3 is H or Y, X4 is H, G, or P, X5 is R or H, X6 is Y, I, or V, X7 is F or H, and X8 is V or H, and optionally, at least one of X1, X2, X3, X4, X5, X7, and X8 is H. (SEQ ID NO: 596); a CDRH2-binding domain comprising a consensus motif, the consensus motif comprising the sequence WIDLENANTIYDAKFQG (SEQ ID NO: 9) or the sequence WIDAGTGX1TX2YSQKFQG, where X1 is L, F, N, or A, and X2 is T or K (SEQ ID NO: 595); a CDRH1-binding domain comprising a consensus motif, the consensus motif comprising the sequence FNIKDYYMH (SEQ ID NO: 47) or the sequence YTFX1X2YX3MH, where wherein X1 is E, S, or T, X2 is S or D, and X3 is A or D (SEQ ID NO: 31); a CDRL3 binding domain comprising the consensus motif comprising the sequence X1QSX2X3X4RT, wherein X1 is K or H, X2 is Y or H, X3 is S, H, L, V, or K, and X4 is H, R, or E, and optionally, at least one of X1, X2, X3, and X4 is H (SEQ ID NO: 598). a CDRH2-binding domain, wherein the consensus motif comprises the WASTRES sequence (SEQ ID NO: 37); a CDRL1-binding domain comprising a consensus motif, wherein the consensus motif comprises the KSSQSLLX1AX2X3X4X5NX6LA sequence, wherein X1 is N or H, X2 is R or H, X3 is T or E, X4 is G or H, X5 is H or K, and X6 is H or Y; and optionally, at least one of X1, X2, X4, X5, and X6 isH (SEQ ID NO: 597). In some embodiments, the antibody or antigen-binding fragment is designated as a Group 2 binder, comprising a CD3-binding domain selected from ADI-48588, ADI-48587, ADI-48577, ADI-48590, ADI-48581, ADI-48575, ADI-48593, ADI-48591, ADI-48647, ADI-48636, ADI-48586, ADI-48646, ADI-48638, ADI-48597, ADI-48601, ADI-48576, ADI-48643, ADI-48624, ADI-48632, ADI-48635, and ADI-48645.

[0248] material and method In addition to the above description, the following materials and methods were employed in this example.

[0249] Preparation of Hu and Cy CD3εδFc heterodimeric antigens. Recombinant heterodimeric CD3 Fc fusion antigens were produced in HEK293 cells by cotransfection with plasmids encoding Hu CD3εFc (extracellular domain, ECD, residues 22–126) and CD3δFc-HIS (ECD residues 22–100) or Cy CD3εFc (ECD residues 22–117) and CD3δFc-HIS (ECD residues 22–100) using heterologous signal peptide sequences. Chromatographic separation was performed on a computer-controlled AKTA Avant 150 preparative chromatography system (GE Healthcare Life Sciences) equipped with an integrated conductivity sensor, allowing in-line salt concentration monitoring during operation. Clarified culture supernatants were purified with Ni Sepharose 6 Fast Flow (GE Healthcare Life Sciences) to remove CD3εεFc-HIS homodimers. CD3εδFc-HIS heterodimers were separated from CD3δδFc-HIS homodimers by Mono Q 10 / 100 GL with a linear Tris-buffered KCl gradient at pH 8.5.

[0250] Peptides. C-terminally biotinylated CD3ε N-terminal peptides were obtained from New England Peptide. All peptides were delivered with a purity of 95% or higher. Peptides were designed based on the primary sequence of Hu CD3ε and the crystal structure of Hu CD3εδ bound to OKT3 (Kjer-Nielsen L. et al. PNAS 2004). The CD3ε N27 peptide has the sequence H2N-QDGNEEMGSITQTPYQVSISGTTVILT[K / SCBiot(dPEG4)]-amide (SEQ ID NO: 56), and the CD3ε N13 peptide has the sequence H2N-QDGNEEMGGITQT[K / SCBiot(dPEG4)]-amide (SEQ ID NO: 57).

[0251] Antigen biotinylation. CD3 antigen was biotinylated using Pierce's EZ-Link Sulfo-NHS-Biotinylation Kit. Goat anti-human F(ab')2 kappa-FITC (LC-FITC), extravidin-PE (EA-PE), and streptavidin-633 (SA-633) were obtained from Southern Biotech, Sigma, and Molecular Probes, respectively. Streptavidin microbeads and MACS LC separation columns were purchased from Miltenyi Biotec.

[0252] Cell line expansion and cell labeling assay. Human Jurkat CD3+ cells (ATCC TIB-152) and Jurkat CD3- cells (ATCC TIB-153) were obtained from ATCC. Cyno HSC-F cells were obtained from the NIH Non-human Primate Reagent Resource. All cell lines were cultured in RPMI 1640 GlutaMax medium supplemented with 10% fetal bovine serum (FBS).

[0253] Cell labeling was performed by dispensing 100,000–200,000 cells per well in a 96-well assay plate. Cells were centrifuged at 500 × g for 5 minutes at 4°C. Cells were then resuspended in 100 μl of 100 nM IgG1 and incubated at room temperature for 20 minutes. Cells were then washed three times with buffer (phosphate-buffered saline (PBS) / 0.1% bovine serum albumin (BSA)) and resuspended in a secondary reagent, typically goat anti-human R-PE (Southern Biotech). Plates were analyzed on a FACSCanto (BD Biosciences) using the HTS sample injector. Flow cytometry data were analyzed relative to the median fluorescence intensity in the R-PE channel.

[0254] FACS affinity pressure selection method. Briefly, yeast cells (at least approximately 2 x 10 7 Cells (labeling condition) were incubated with a sufficient amount of biotinylated antigen to provide a stoichiometric excess relative to the average IgG presentation. Antigen labeling conditions were 100–1 nM under equilibrated conditions, typically performed for 20 minutes to several hours at room temperature in FACS wash buffer (phosphate-buffered saline (PBS) / 0.1% bovine serum albumin (BSA)). After three washes with wash buffer, yeast were stained for 15 minutes at 4°C with a 1:100 dilution of the secondary reagent anti-human light chain FITC conjugate (LC-FITC) and either a 1:500 dilution of streptavidin-633 (SA-633) or a 1:50 dilution of extraavidin-phycoerythrin (EA-PA). After two washes with ice-cold wash buffer, the cell pellet was collected, typically at 1 x 10 7 The yeast were resuspended in at least 1 mL of wash buffer per yeast and transferred to a strainer-capped sort tube. Sorting was performed using a FACS ARIA sorter (BD Biosciences) to determine sort gating and select binders. After the final round of sorting, the yeast were plated and individual colonies were picked and characterized.

[0255] Yeast production and purification of antibodies. Yeast clones were grown to saturation and then induced for 48 hours at 30°C with shaking. After induction, yeast cells were pelleted and the supernatant was collected for purification. IgG was purified using a Protein A column and eluted with acetic acid at pH 2.0. Fab fragments were generated by papain digestion and purified with KappaSelect or CaptureSelect IgG-CH1 (GE Healthcare LifeSciences).

[0256] HEK Production and Purification of Antibodies. Mammalian expression of IgG was achieved by subcloning the antibody into a new expression vector, followed by transient transfection and expression in HEK293ADI1. A monoclonal cell line derived from HEK293 (DSMZ) was selected for its aggregation-free growth, growth rate, and transfectability. Briefly, the expression vector containing the antibody of interest was transfected by complexation with a transfection reagent, followed by exposure to HEK cells for 1 hour, followed by dilution in culture medium to a final density of 4 million cells per mL. The cells were then cultured for 7 days, with fresh feed medium changes every 48 hours. After 7 days, the supernatant was collected after centrifugation and purified using Protein A. Where necessary, CHT column purification was added to achieve >95% monomericity.

[0257] Cell binding assay. CD3+ human Jurkat cells (ATCC) and CHO-S cells (Invitrogen / ThermoFisher) were lysed and washed with chilled PBSF buffer, pH 7.4 (PBS + 0.1% BSA, pH 7.4). Approximately 200,000 cells were dispensed per well of a 96-well plate (FACS Assay Plate VWR BD 353263) and pelleted by centrifugation (500 x g for 5 minutes). Cells were washed with either PBSF pH 7.4 or PBSF pH 6.0 (PBS + 0.1% BSA, pH 6.0) and then resuspended in 100 μl of either PBSF pH 7.4 or PBSF pH 6.0 containing IgG antibody (100 nM) produced in yeast as described above. The mixture (cells + antibody) was incubated on ice for 20 minutes and then washed twice with either PBSF pH 7.4 or PBSF pH 6.0. Cells were resuspended in 50 μl of propidium iodide (Roche, 1:500 dilution) and anti-human IgG-RPE (Southern Biotech, 1:100 dilution) prepared in either PBSF pH 7.4 or PBSF pH 6.0. After incubation on ice in the dark for 20 minutes, cells were washed twice with either PBSF pH 7.4 or PBSF pH 6.0. Binding was analyzed on a FACS Canto II.

[0258] ForteBio KD Measurement (BioLayer Interferometry; BLI). ForteBio affinity measurements were generally performed as previously reported (Estep, P., et al., High throughput solution-based measurement of antibody-antigen affinity and epitope binning. MAbs, 2013.5(2):p.270-8). Briefly, ForteBio affinity measurements were performed by online loading of IgG onto the AHC sensor. The sensor was equilibrated offline in assay buffer for 30 minutes, followed by online monitoring for 60 seconds to establish a baseline. The IgG-loaded sensors were exposed to 100 nM antigen (e.g., CD3) for 5 minutes, then transferred to assay buffer for 5 minutes, and dissociation rate measurements were performed. A 1:1 binding model was used to analyze the kinetics.

[0259] PSR Preparation. The polyspecific reaction reagent (PSR) was prepared as described, for example, in WO2014 / 179363 and Xu et al., Protein Eng Des Sel, 26(10):663-670 (2013). Briefly, 2.5 liters of CHO-S cells were used as starting material. In a 500 mL centrifuge bottle filled to 400 mL, the cells were pelleted at 2,400 x g for 5 minutes. The cell pellets were combined and then resuspended in 25 mL of Buffer B and pelleted at 2,400 x g for 3 minutes. The buffer was decanted, and the wash was repeated once more. The cell pellets were resuspended in 3x the pellet volume of Buffer B containing 1x protease inhibitor (Roche, Complete, EDTA-free) using a Polytron homogenizer while keeping the cells on ice. The homogenate was then centrifuged at 2,400 × g for 5 minutes, and the supernatant was retained and pelleted once more (2,400 × g / 5 minutes) to ensure removal of unbroken cells, cell debris, and nuclei. The resulting supernatant was the total protein preparation. The supernatant was then transferred to two Nalgene Oak Ridge 45 mL centrifuge tubes and pelleted at 40,000 × g for 40 minutes at 4°C. The supernatant containing the separated cytosolic proteins (SCP) was then transferred to a clean Oak Ridge tube and centrifuged once more at 40,000 × g. In parallel, the pellet containing the membrane fraction (EMF) was retained and centrifuged at 40,000 × g for 20 minutes to remove any remaining supernatant. The EMF pellet was then rinsed with Buffer B. 8 mL of Buffer B was then added to the membrane pellet, which was then scraped and transferred to a Dounce homogenizer. After the pellets were homogenized, they were transferred to a 50 mL conical tube to represent the final EMF preparation.

[0260] about 10 6 ~10 7One billion cells / mL of mammalian cells (e.g., CHO, HEK293, Sf9, etc.) were transferred from tissue culture medium into four 250 mL conical tubes and pelleted at 550 x g for 3 minutes. All subsequent steps were performed at 4 °C or on ice using ice-cold buffers. The cells were washed with 100 mL of PBSF (1x PBS + 1 mg / mL BSA) and combined in one conical tube. After removing the supernatant, the cell pellet was resuspended in 30 mL of buffer B (50 mM HEPES, 0.15 M NaCl, 2 mM CaCl, 5 mM KCl, 5 mM HCl). The cells were resuspended in 10% MgCl2, 10% glycerol, pH 7.2, and pelleted at 550 x g for 3 min. The supernatant in Buffer B was decanted, and the cells were resuspended in 3x the pellet volume of Buffer B supplemented with 2.5x protease inhibitors (Roche, cOmplete, EDTA-free). From this point on, protease inhibitors were included in Buffer B. The cells were homogenized (Polyton homogenizer, PT1200E) with four 30-second pulses, and the membrane fraction was pelleted at 40,000 x g for 1 h at 4°C. The pellet was collected in 1 mL of PBS. The pellet was transferred to a Dounce homogenizer with 3 mL of buffer B and resuspended by slowly moving the pestle up and down 30-35 times. The enriched membrane fraction (EMF) was transferred to a new collection tube, and the pestle was rinsed to collect all potential proteins. The protein concentration of the purified EMF was determined using a Dc-protein assay kit (BioRad). To solubilize the EMF, solubilization buffer (50 mM NaCl) was added. The mixture was transferred to a final concentration of 1 mg / mL in 50 mL of PBS containing 1% HEPES, 0.15 M NaCl, 2 mM CaCl, 5 mM KCl, 5 mM MgCl, 1% n-dodecyl-bD-maltopyranoside (DDM), 1x protease inhibitors, pH 7.2. The mixture was rotated overnight at 4°C and then centrifuged at 40,000 x g for 1 hour in a 50 mL Oak Ridge tube (Fisher Scientific, 050529-ID). The supernatant, which contains soluble membrane protein (SMP), was collected and protein production was quantified as described above.

[0261] For biotinylation, prepare NHS-LC-biotin stock solution according to the manufacturer's protocol (Pierce, Thermo Fisher). Briefly, 20 μl of biotin reagent was added to every 1 mg of EMF sample and incubated at 4°C for 3 hours with gentle agitation. Adjust the volume to 25 mL with Buffer B and transfer to an Oak Ridge centrifuge tube. Precipitate biotinylated EMF (b-EMF) at 40,000 × g for 1 hour and rinse twice with 3 mL of Buffer C (Buffer B minus glycerol) without disturbing the pellet. Remove residual solution. Resuspend the pellet in 3 mL of Buffer C using a Dounce homogenizer as described above. The resuspended pellet is now biotinylated EMF (b-EMF). It was solubilized as described above for preparing b-SMP.

[0262] PSR binding analysis. Assays were generally performed as described, for example, in Xu et al., Protein Eng Des Sel, 26(10):663-670 (2013). To characterize the PSR profile of monoclonal antibodies displayed on yeast, 2 million IgG-displaying yeast cells were transferred to a 96-well assay plate and pelleted at 3000 x g for 3 minutes. The supernatant was removed. The pellet was resuspended in 50 μl of a 1:10 dilution of freshly prepared stock b-PSR and incubated on ice for 20 minutes. The cells were washed twice with 200 μl of ice-cold PBSF and resuspended in 50 μl of secondary labeling mix (Extravidin-R-PE, anti-human LC-FITC, and propidium iodide). The mixture was incubated on ice for 20 minutes, followed by two washes with 200 μl of ice-cold PBSF. Cells were resuspended in 100 μl of ice-cold PBSF and plated on a FACSCanto (BD Biosciences) using the HTS sample injector. Flow cytometry data were analyzed for mean fluorescence intensity in the R-PE channel and normalized to appropriate controls for assessment of nonspecific binding. Numerous methods for displaying or displaying antibodies or antibody fragments on the surface of yeast have been previously described, all of which are compatible with the present protocol (Blaise et al., Gene, 342(2):211-8(2004); Boder and Wittrup, Nat Biotechnol., 15(6):553-7(1997); Kuroda and Ueda, Biotechnol Lett., 33(1):1-9(2011); Orcutt and Wittrup, Springer Protocols: Antibody Engineering, 1:207-233(2010); Rakestraw et al., Protein Eng Des Sel., 24(6):525-30(2011); Sazinsky et al., Proc Natl Acad Sci U S A., 105(51):20167-72(2008); Tasumi et al., Proc Natl Acad Sci US A., 106(31):12891-6(2009)).

[0263] ForteBio Kinetics. A ForteBio Octet HTX instrument was used in 12-channel mode (8 sensors per channel, 96 sensors per experiment) with either AHC, SA, or AHQ sensors. The instrument was operated with manufacturer-supplied software (versions 8.2 and 9.0). Sample names and concentrations were entered on the plate data page, and proteins bound to the sensors were identified in the "Information" column on the sensor data page. Kinetic experiments were collected with either a 90- or 180-second baseline, a 180-second association phase, and a 180-second dissociation phase. All files were saved to a shared network drive using a naming convention that identifies the experiment type.

[0264] HIC. The IgG1 sample was buffer exchanged into 1 M ammonium sulfate and 0.1 M sodium phosphate at pH 6.5 using a Zeba 40 kDa 0.5 mL spin column (Thermo Pierce, catalog number 87766). A salt gradient was established on a Dionex ProPac HIC-10 column from 1.8 M ammonium sulfate and 0.1 M sodium phosphate at pH 6.5 to the same conditions without ammonium sulfate. The gradient was run for 17 min at a flow rate of 0.75 ml / min. An acetonitrile wash step was added at the end of the run to remove any remaining protein, and the column was re-equilibrated for over 7 column volumes before the next injection cycle. Peak retention times were monitored by absorbance at A280, and the ammonium sulfate concentration at elution was calculated based on the gradient and flow rate.

[0265] LCMS. The mAb sample was reduced with DTT and then subjected to mid-down LCMS analysis on a Bruker maXis 4G mass spectrometer coupled to an Agilent 1100 HPLC (Agilent). A POROS R2 10 μm (2.1 x 30 mm) reversed-phase column was used to remove salts from the sample. A fast LC flow of 2 mL / min allowed for separation of the sample and salt, and sample elution and column regeneration were completed within a 2.1-minute cycle. A T-junction was used to deliver a sample flow of only 0.15 mL / min to the mass spectrometer for sample analysis. The Bruker maXis 4G mass spectrometer was operated in positive ion mode with detection in the 750–2500 m / z range. The remaining source parameters were set as follows: the capillary was set at 5500 V, the nebulizer at 4.0 bar, the drying gas at 4.0 L / min, and the drying temperature at 200 °C.

[0266] MS spectra were analyzed using Bruker Data Analysis version 4.1, and deconvolution was performed using maximum entropy deconvolution in the mass range of 20–30 kDa.

[0267] A concise sequence listing is provided below in Table 1. The concise sequence listing provides the amino acid sequence of the heavy chain variable region (HC), with each of the heavy chain variable region CDRs underlined. The amino acid sequence of the light chain variable region (LC) is also provided, with each of the light chain variable region CDRs underlined.

[0268] [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4]

Table 1-5

Table 1-6

Table 1-7

Table 1-8

Table 1-9

Table 1-10

Table 1-11

Table 1-12

Table 1-13

Table 1-14

Table 1-15

Table 1-16

Table 1-17

Table 1-18

Table 1-19

Table 1-20

Table 1-21

Table 1-22

Table 1-23

Table 1-24

Table 1-25

Table 1-26

Table 1-27

Table 1-28

Table 1-29

Table 1-30

Table 1-31

Table 1-32

Table 1-33

Table 1-34

Table 1-35

Table 2-1

Table 2-2

Table 2-3

Table 2-4

Table 2-5

Table 2-6

Table 2-7

Table 2-8

Table 2-9

Table 2-10

Table 2-11

Table 2-12

Table 2-13

Table 2-14

Table 2-15

Table 2-16

Table 2-17

Table 3-1

Table 3-2

Table 3-3

Table 3-4

Table 4

Claims

1. Anti-cluster of differentiation 3 (CD3) antibodies and / or antigen-binding fragments, (a) i. AX １ DX ２ X ３ X ４ X ５ X ６ X ７ X ８ DX ９ And X １ is R or H, and X ２ is A, H, M or Q, and X ３ is Y, H, S, G, A, T, V or R, and X ４ is G, H, P, E or R, and X ５ is H or R, and X ６ is Y, N, F, H, D, E, S, L, M, I, G, A, Q or T, and X ７ is F or H, and X ８ is Y or H, and X ９ is V, H, or M, and optionally, X １ , X ２ , X ３ , X ４ , X ５ , X ６ , X ７ , X ８ and X ９ wherein at least one of the following is H (SEQ ID NO: 58); ii. ARDX １ X ２ X ３ X ４ YFYDX ５ And X １ is H or A, and X ２ is T, Y or H, and X ３ is G or H, and X ４ is H, R, V or I, and X ５ is V or H, and optionally, X １ , X ２ , X ３ , X ４ and X ５ wherein at least one of the following is H (SEQ ID NO: 43); iii. AX １ DX ２ YX ３ HX ４ FYDV, X １ is R or H, and X ２ is A or H, and X ３ is G, H or P, and X ４ is Y, H, D, V, E, S, N, L, M, I, G, A, Q or T, and optionally, X １ , X ２ , X ３ , and X ４ wherein at least one of is H (SEQ ID NO: 1); iv. ARDX １ YGX ２ X ３ X ４ YDX ５ And X １ is A or H, and X ２ is R or H, and X ３ is H or Y, and X ４ is F or H, and X ５ is H or V, and optionally, X １ , X ２ , X ３ , X ４ , and X ５ wherein at least one of the following is H (SEQ ID NO: 2); v. ARDAHX １ X ２ YX ３ X ４ DX ５ And X １ is G, E or R, and X ２ is R or H, and X ３ is F or H, and X ４ is Y or H, and X ５ is V or H, and optionally, X １ , X ２ , X ３ , X ４ and X ５ wherein at least one of the following is H (SEQ ID NO: 3); vi. ARDAX １ HRX ２ FYDV, X １ is H, Y, S, G, A, T, V or R, and X ２ is Y or H, and optionally, X １ and X ２ wherein at least one of the following is H (SEQ ID NO: 4); vii. ARDX １ YHRYFYDX ２ And X １ is H or A, and X ２ is H, V or M, and optionally, X １ and X ２ wherein at least one of the following is H (SEQ ID NO: 5); viii. AX １ DAYX ２ X ３ X ４ HX ５ Domestic violence, X １ is R or H, and X ２ is G or H, and X ３ is H or R, and X ４ is N, F or Y, and X ５ is Y or H, and optionally, X １ , X ２ , X ３ , X ４ and X ５ wherein at least one of the following is H (SEQ ID NO: 6); IX. ARDX １ X ２ GRYFYDV and X １ is M, Q or H, and X ２ is R or H, and optionally, X １ and X ２ wherein at least one of the following is H (SEQ ID NO: 7); x. ARDX1X2X3RYFYDX4, where X １ is H or A, and X ２ is T, Y or H, and X ３ is G or H, and X ４ is V or H, and optionally, X １ , X ２ , X ３ , and X ４ wherein at least one of the following is H (SEQ ID NO: 8); xi. ARDAX １ X ２ X ３ X ４ FYDX ５ And X １ is T, H or Y, and X ２ is G or H, and X ３ is H or R, and X ４ is V or Y, and X ５ is V or H, and optionally, X １ , X ２ , X ３ and X ５ wherein at least one of the is H (SEQ ID NO: 593), and xii. AX １ DX ２ X ３ X ４ X ５ X ６ X ７ YDX ８ And X １ is R or H, and X ２ is H or A, and X ３ is H or Y, and X ４ is H, G or P, and X ５ is R or H, and X ６ is Y, I or V, and X ７ is F or H, and X ８ is V or H, and optionally, X １ , X ２ , X ３ , X ４ , X ５ , X ７ and X ８ and wherein at least one of the V is H (SEQ ID NO: 596). Ｈ Variable heavy chain (VH) containing CDR3 (CDRH3) Ｈ ) chain polypeptide, (b) i.X １ IX ２ X ３ X ４ X ５ X ６ X ７ TX ８ YSQKFQG, where X １ is W, S, Y, F, G or D, and X ２ is N, T, D, V or H, and X ３ is A, P or S, and X ４ is G, A, S, N, D, L, V, H, Q, T, I or Y, and X ５ is D or T, and X ６ is A or G, and X ７ is A, N, T, S, L, D, F, Y or E, and X ８ is V, K, T, D, Y, F, A, H, N, L, I or E, and optionally X １ , X ２ , X ３ , X ４ , X ５ , X ６ , X ７ and X ８ wherein at least one of the following is H (SEQ ID NO: 59); ii. WIDLENANTIYDAKFQG (SEQ ID NO: 9), iii. X １ INPX ２ TGX ３ TX ４ YSQKFQG, where X １ is W or Y, and X ２ is A, S, D, G, N, L, V, H or Q, and X ３ is A, T or S, and X ４ is K, V, T, D, Y, F or A (SEQ ID NO: 10); iv. X １ IX ２ AGTGX ３ TX ４ YSQKFQG, where X １ is W, Y or F, and X ２ is T, N or D, and X ３ is A, T or L, and X ４ is A, K, V, H, T or N (SEQ ID NO: 11); v. X １ IDAGTGX ２ TX ３ YSQKFQG, where X １ is S or W, and X ２ is L, N, D or F, and X ３ is D, Y or K (SEQ ID NO: 12); vi. X １ IX ２ AGTGATX ３ YSQKFQG, where X １ is G, D or S, and X ２ is I or D, X ３ is K or D (SEQ ID NO: 13); vii. WINPX １ TGNTX ２ YSQKFQG, where X １ is D, T, L, S or A, and X ２ is D, V, L or N (SEQ ID NO: 14); viii. X １ INAGTGX ２ TX ３ YSQKFQG, where X １ is Y or W, and X ２ is N, D or A, and X ３ is I or V (SEQ ID NO: 15); ix. X １ INPX ２ TGX ３ TKYSQKFQG, and X １ is W or Y, and X ２ is D, I or Y, and X ３ is D, Y or E (SEQ ID NO: 16); x.SIX １ AGTGX ２ TKYSQKFQG, and X １ is N or V, and X ２ is A or I (SEQ ID NO: 17); xi. SINAGTGX １ TX ２ YSQKFQG, where X １ is F or N, and X ２ is Y or D (SEQ ID NO: 18); xii. X １ IX ２ X ３ GTGX ４ TDYSQKFQG, where X １ is D or W, and X ２ is N or H, and X ３ is A or S, and X ４ is A or N (SEQ ID NO: 19); xiii. WIDPX １ TGATX ２ YSQKFQG, where X １ is N, H or Y, and X ２ is V or K (SEQ ID NO: 20); xiv. WIX １ PX ２ TGNTKYSQKFQG, where X １ is D or N, and X ２ is L, I or V (SEQ ID NO: 21); xv. SINAGDANTKYSQKFQG (SEQ ID NO: 22), xvi. X １ IDPX ２ TGATX ３ YSQKFQG, where X １ is D or W, and X ２ is D or V, and X ３ is E or D (SEQ ID NO: 23); xvii. WINAGDAATVYSQKFQG (SEQ ID NO: 24), and xviii. WIDAGTGX １ TX ２ YSQKFQG, where X １ is L, F, N or A, and X ２ is selected from the group consisting of: T or K (SEQ ID NO: 595) Ｈ Variable-heavy (VH) containing chain CDR2 (CDRH2) Ｈ ) chain polypeptide, (c) i. FNIKDYHMH (SEQ ID NO: 25), ii. SNIKDYYMH (SEQ ID NO: 26); iii. SNIKDYHMH (SEQ ID NO: 27); iv. YTFX １ X ２ X ３ X ４ MH and X １ is A, K, D, Q, E, N, T, L, Y, S, P, G, H or V, and X ２ is T, S or A, and X ３ is Y or I, and X ４ is A, D, N, S, Y, T, I, V, L, E, P, R or G (SEQ ID NO: 28); v. YTFX １ X ２ X ３ X ４ MH and X １ is T, D, A, N or V, and X ２ is D, E, G or Q, and X ３ is Y or D, and X ４ is D, A, E, N, S, Y or V (SEQ ID NO: 29); vi. YTFTSX １ X ２ MH and X １ is A, D or T, and X ２ is D, F, A, M, V or Y (SEQ ID NO: 30); vii. YTFX １ X ２ YX ３ MH and X １ is N or T, and X ２ is Q or N, and X ３ is S, T or A (SEQ ID NO: 31), and viii. YTFX １ X ２ YVMH, and X １ is I or N, and X ２ is K or R (SEQ ID NO: 32); ix. FNIKDYYMH (SEQ ID NO: 47), and x. YTFX １ X ２ YX ３ MH and X １ is E, S or T, and X ２ is S or D, and X ３ is selected from the group consisting of A or D (SEQ ID NO: 31) Ｈ Variable-heavy (V) Ｈ ) chain polypeptide, (d) i.X １ X ２ SX ３ X ４ X ５ RX ６ And X １ is H, K or G, and X ２ is Q or H, and X ３ is Y or H, and X ４ is S, H, D, T, V, M or L, and X ５ is R or H, and X ６ is T or H, and optionally, X １ , X ２ , X ３ , X ４ , X ５ and X ６ wherein at least one of the following is H (SEQ ID NO: 33); ii. KQSYX １ X ２ RT and X １ is H, V, K, W, R, L, G, Y or Q, and X ２ is H, L, E, W, G, M, P, T, Q or V, and optionally, X １ and X ２ wherein at least one of the following is H (SEQ ID NO: 34); iii. X １ QSX ２ HX ３ RT and X １ is K or H, and X ２ is H, Y, M, S, L, E, G or W, and X ３ is R or K, and optionally, X １ and X ２ wherein at least one of the following is H (SEQ ID NO: 35); iv. KQSX １ X ２ X ３ RT and X １ is Y or H, and X ２ is T, S, V or K, and X ３ is R or H, and optionally, X １ and X ３ wherein at least one of the following is H (SEQ ID NO: 36); v. KQSX １ X ２ X ３ RT and X １ is H or Y, and X ２ is T, S or Q, and X ３ is R or H, and optionally, X １ and X ３ wherein at least one of is H (SEQ ID NO: 36), and vi. X １ QSX ２ X ３ X ４ RT and X １ is K or H, and X ２ is Y or H, and X ３ is S, H, L, V or K, and X ４ is H, R or E, and optionally, X １ , X ２ , X ３ and X ４ and wherein at least one of the V is H (SEQ ID NO: 598). Ｌ Variable light chain (VLCD) containing CDR3 (CDRL3) Ｌ ) chain polypeptide, (e) V of WASTRES (SEQ ID NO: 37) Ｌ The variable light chain (VLCD) containing CDR2 (CDRL2) Ｌ ) chain polypeptides, and / or (f) i. KSSQSLLX １ X ２ X ３ X ４ GX ５ NX ６ LA, X １ is N or H, and X ２ is A, R or T, and X ３ is R or H, and X ４ is T, P or E, and X ５ is H or K, and X ６ is H or Y, and optionally, X １ , X ３ , X ５ and X ６ wherein at least one of the following is H (SEQ ID NO: 38); ii. KSSQSLLX １ AX ２ THX ３ NX ４ LA, X １ is N or H, and X ２ is R or H, and X ３ is K or H, and X ４ is Y or H, and optionally, X １ , X ２ , X ３ and X ４ wherein at least one of the following is H (SEQ ID NO: 39); iii. KSSQSLLNASTAKNYLA (SEQ ID NO: 40), iv. KSSQSLLNARTRTNYLA (SEQ ID NO: 41), v. KSSQSLLNX １ X ２ X ３ GX ４ NX ５ LA, X １ is S or A, and X ２ is R or H, and X ３ is E or T, and X ４ is H or K, and X ５ is H or Y, and optionally, X ２ , X ４ and X ５ wherein at least one of the following is H (SEQ ID NO: 42); vi. KSSQSLLNX １ X ２ TGX ３ NYLA, X １ is A or S, and X ２ is R or H, and X ３ is H or K, and optionally, X ２ and X ３ wherein at least one of the is H (SEQ ID NO: 594), and vii. KSSQSLLX １ AX ２ X ３ X ４ X ５ NX ６ LA, X １ is N or H, and X ２ is R or H, and X ３ is T or E, and X ４ is G or H, and X ５ is H or K, and X ６ is H or Y, and optionally, X １ , X ２ , X ４ , X ５ and X ６ and wherein at least one of the V is H (SEQ ID NO: 597). Ｌ The variable light chain (VLCD) containing CDR1 (CDRL1) Ｌ ) chain polypeptide.

2. (a) Amino acid sequence ARDX １ X ２ X ３ X ４ YFYDX ５ And X １ is H or A, and X ２ is T, Y or H, and X ３ is G or H, and X ４ is H, R, V or I, and X ５ is V or H, and optionally, X １ , X ２ , X ３ , X ４ and X ５ wherein at least one of: (b) Amino acid sequence KQSX １ X ２ X ３ RT and X １ is Y or H, and X ２ is T, S, V or K, and X ３ is R or H, and optionally, X １ and X ３ wherein at least one of: (c) Amino acid sequence KSSQSLLNX １ X ２ X ３ GX ４ NX ５ LA, X １ is S or A, and X ２ is R or H, and X ３ is E or T, and X ４ is H or K, and X ５ is H or Y, and optionally, X ２ , X ４ and X ５ 2. The anti-CD3 antibody and / or antigen-binding fragment of claim 1, comprising a CDRL1 comprising:

3. (a) Amino acid sequence ARDAX １ X ２ X ３ X ４ FYDX ５ And X １ is T, H or Y, and X ２ is G or H, and X ３ is H or R, and X ４ is V or Y, and X ５ is V or H, and optionally, X １ , X ２ , X ３ and X ５ wherein at least one of: (b) a CDRH2 comprising the amino acid sequence WIDLENANTIYDAKFQG (SEQ ID NO: 9); (c) a CDRH1 comprising the amino acid sequence FNIKDYYMH (SEQ ID NO: 47); (d) amino acid sequence KQSX １ X ２ X ３ RT and X １ is H or Y, and X ２ is T, S or Q, and X ３ is R or H, and optionally, X １ and X ３ wherein at least one of: (e) a CDRL2 comprising the amino acid sequence WASTRES (SEQ ID NO: 37), and / or (f) Amino acid sequence KSSQSLLNX １ X ２ TGX ３ NYLA, X １ is A or S, and X ２ is R or H, and X ３ is H or K, and optionally, X ２ and X ３ and a CDRL1 comprising at least one of:

4. (a) amino acid sequence AX １ DX ２ X ３ X ４ X ５ X ６ X ７ YDX ８ And X １ is R or H, and X ２ is H or A, and X ３ is H or Y, and X ４ is H, G or P, and X ５ is R or H, and X ６ is Y, I or V, and X ７ is F or H, and X ８ is V or H, and optionally, X １ , X ２ , X ３ , X ４ , X ５ , X ７ and X ８ wherein at least one of: (b) i. WIDLENANTIYDAKFQG (SEQ ID NO: 9), and ii. WIDAGTGX １ TX ２ YSQKFQG, where X １ is L, F, N or A, and X ２ is T or K (SEQ ID NO:595); (c) i. FNIKDYYMH (SEQ ID NO: 47), and ii. YTFX １ X ２ YX ３ MH and X １ is E, S or T, and X ２ is S or D, and X ３ is A or D (SEQ ID NO: 31); (d) amino acid sequence X １ QSX ２ X ３ X ４ RT and X １ is K or H, and X ２ is Y or H, and X ３ is S, H, L, V or K, and X ４ is H, R or E, and optionally, X １ , X ２ , X ３ and X ４ wherein at least one of: (e) a CDRL2 comprising the amino acid sequence WASTRES (SEQ ID NO: 37); and / or (f) Amino acid sequence KSSQSLLX １ AX ２ X ３ X ４ X ５ NX ６ LA, X １ is N or H, and X ２ is R or H, and X ３ is T or E, and X ４ is G or H, and X ５ is H or K, and X ６ is H or Y, and optionally, X １ , X ２ , X ４ , X ５ and X ６ and a CDRL1 comprising at least one of:

5. (a) the antibody or antigen-binding fragment, while eliciting T cell activation or T cell killing, exhibits a reduced tendency to elicit cytokine production to levels capable of inducing cytokine release syndrome; (b) the antibody or antigen-binding fragment comprises a multispecific antibody; (c) the antibody or antigen-binding fragment comprises a bispecific antibody; (d) the antibody or antigen-binding fragment comprises an scFv; (e) the antibody or antigen-binding fragment comprises at least a second antigen-binding domain that specifically binds to a tumor target, an immune-tumor target, a neurodegenerative disease target, an autoimmune disorder target, an infectious disease target, a metabolic disease target, a cognitive disorder target, a blood-brain barrier target, or a hematological disorder target; (f) the antibody or antigen-binding fragment is selected from the group consisting of 17-IA, 4-1BB, 4Dc, 6-keto-PGF1a, 8-iso-PGF2a, 8-oxo-dG, Al adenosine receptor, A33, ACE, ACE-2, activin, activin A, activin AB, activin B, activin C, activin RIA, activin RIA ALK-2, and activin RIB ALK-4, activin RIIA, activin RUB, ADAM, ADAM10, ADAM12, ADAM15, ADAM17 / TACE, ADAM8, ADAM9, ADAMTS, ADAMTS4, ADAMTS5, addressin, aFGF, ALCAM, ALK, ALK-1, ALK-7, alpha-l-antitrypsin, alpha-V / beta-1 antagonist, ANG, Ang, APAF-1, APE, APJ, APP, APRIL, AR, ARC, ART, artemin, anti-Id, ASPARTIC, atrial natriuretic factor, av / b3 Integrin, Axl, b2M, B7-1, B7-2, B7-H, B-lymphocyte stimulatory factor (BlyS), BACE, BACE-1, Bad, BAFF, BAFF-R, Bag-1, BAK, Bax, BCA-1, BCAM, Bel, BCMA, BDNF, b-ECGF, bFGF, BID, Bik, BFM, BLC, BL-CAM, BLK, BMP, BMP-2 BMP-2a, BMP-3 osteogenin, BMP-4 BMP-2b, BMP-5, BMP-6 Vgr-1, BMP-7 (OP-1), BMP-8 (BMP-8a, OP-2), BMPR, BMPR-IA (ALK-3), BMPR-IB (ALK-6), BRK-2, RPK-1, BMPR-II (BRK-3), BMPs, b-NGF, BOK, bombesin, bone-derived neurotrophic factor, BPDE, BPDE-DNA, BTC, complement factor 3 (C3), C3a, C4, C5, C5a, C IO, CA125, CAD-8, calcitonin, cAMP, carcinoembryonic antigen (CEA), tumor-associated antigen, cathepsin A, cathepsin B, cathepsin C / DPPI, cathepsin D, cathepsin E, cathepsin H, cathepsin L, cathepsin O, cathepsin S, cathepsin V, cathepsin X / Z / P, CBL, CCI, CCK2, CCL, CCL1, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL2, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9 / 10, CCR, CCR1, CCR10, CCR10, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CD1, CD2, CD4, CD5, CD6, CD7, CD8, CD10, CDlla, CDllb, CDllc, CD13, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD27L, CD28, CD29, CD30, CD30L, CD32, CD33 (p67 protein), CD34, CD38, CD40, CD40L, CD44, CD45, CD46, CD49a, CD52, CD54, CD55, CD56, CD61, CD64, CD66e, CD74, CD80 (B7-1), CD89, CD95, CD123, CD137, CD138, CD140a, CD146, CD147, CD148, CD152, CD164, CEACAM5, CFTR, cGMP, CINC, Botulinum toxin, Welch bacillus toxin, CKb8-1, CLC, CMV, CMV UL, CNTF, CNTN-1, COX, C-Ret, CRG-2, CT-1, CTACK, CTGF, CTLA-4, CX3CL1, CX3CR1, CXCL, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCR, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, Cytokeratin tumor-associated antigen, DAN, DCC, DcR3, DC-SIGN, Decay accelerating factor, des(1-3)-IGF-I (Brain IGF-1), Dhh, Digoxin, DNAM-1, Dnase, Dpp, DPPIV / CD26, Dtk, E-cad, EDA, EDA-A1, EDA-A2, EDAR, EGF, EGFR (ErbB-1), EMA, EMMPRIN, EN A, Endothelin receptor, Enkephalinase, eNOS, Eot, eotaxin1, EpCAM, Ephrin B2 / EphB4, EPO, ERCC, E-selectin, ET-1, Factor Ila, Factor VII, Factor VIIIc, Factor IX, fibroblast activation protein (FAP), Fas, FcRl, FEN-1, ferritin, FGF, FGF-19, FGF-2, FGF3, FGF-8, FGFR, FGFR-3, fibrin, FL, FLIP, Flt-3, Flt-4, follicle-stimulating hormone, fractalkine, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10, G250, Gas6, GCP-2, GCSF, GD2, GD3, GDF, GDF-1, GDF-3 (Vgr-2), GDF-5 (BMP-14, CDMP- 1), GDF-6 (BMP-13, CDMP-2), GDF-7 (BMP-12, CDMP-3), GDF-8 (myostatin), GDF-9, GDF-15 (MIC-1), GDNF, GFAP, GFRα-1, GFR-alpha1, GFR-alpha2, GFR-alpha3, GITR, glucagon, Glut 4, glycoprotein Ilb / IIIa (GP Ilb / IIIa), GM-CSF, gpl30, gp72, GRO, growth hormone-releasing factor, hapten (NP-cap or NIP-cap), HB-EGF, HCC, HCMV gB envelope glycoprotein, HCMV gH envelope glycoprotein, HCMV UL, hematopoietic growth factor (HGF) growth factor), Hep B gpl20, heparanase, Her2, Her2 / neu (ErbB-2), Her3 (ErbB-3), Her4 (ErbB-4), herpes simplex virus (HSV) gB glycoprotein, HSV gD glycoprotein, HGFA, high molecular weight melanoma-associated antigen (HMW-MAA), HIV gpl20, HIV IIIB gp 120 V3 loop, HLA, HLA-DR, HM1.24, HMFGPEM, HRG, Hrk, human cardiac myosin, human cytomegalovirus (HCMV), human growth hormone (HGH), HVEM, 1-309, IAP, ICAM, ICAM-1, ICAM-3, ICE, ICOS, IFNg, Ig, IgA receptor, IgE, IGF, IGF-binding protein, IGF-1R, IGFBP, IGF-I, IGF-II, IL, IL-1, IL- 1R, IL-2, IL-2R, IL-4, IL-4R, IL-5, IL-5R, IL-6, IL-6R, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-18, IL-18R, IL-23, interferon (INF)-alpha, INF-beta, INF-gamma, inhibin, iNOS, insulin A-chain, insulin B-chain, insulin Integrin-like growth factor 1, integrin alpha 2, integrin alpha 3, integrin alpha 4, integrin alpha 4 / beta 1, integrin, alpha 4 / beta 7, integrin alpha 5 (alpha V), integrin alpha 5 / beta 1, integrin alpha 5 / beta 3, integrin alpha 6, integrin beta 1, integrin beta 2, interferon gamma, IP-10, 1-TAC, JE, kallikrein 2, kallikrein 5, kallikrein 6, kallikrein 11, kallikrein 12, kallikrein 14, kallikrein 15, kallikrein LI, kallikrein L2, kallikrein L3, kallikrein L4, KC, KDR, keratinocyte growth factor (KGF), laminin 5, LAMP, LAP, LAP (TGF-1), latent TGF-1, latent TGF-1bpl, LBP, LDGF, LECT2, Lefty, Lewis-Y antigen, Lewis-Y related antigen, LFA-1, LFA-3, Lfo, LIF, LIGHT, lipoprotein, LIX, LKN, Lptn, L-selectin, LT-a, LT-b, LTB4, LTBP-1, pulmonary surfactant, progesterone, lymphotoxin beta receptor, Mac-1, MAdCAM, MAG, MAP2, M ARC, MCAM, MCAM, MCK-2, MCP, M-CSF, MDC, Mer, metalloproteinase, MGDF receptor, MGMT, MHC (HLA-DR), MIF, MIG, MIP, MIP-1-alpha, MK, MMAC1, MMP, MMP-1, MMP-10, MMP-11, MMP-12, MMP-13, MMP-14, MMP-15, MMP-2, MMP-24, MMP- 3, MMP-7, MMP-8, MMP-9, MPIF, Mpo, MSK, MSP, mucin (Mucl), MUC18, Müllerian inhibitory factor, Mug, MuSK, NAIP, NAP, NCAD, N-cadherin, NCA90, NCAM, NCAM, neprilysin, neurotrophin-3, -4, or -6, neurturin, nerve growth factor (NGF), NGFR, NGF-beta, nNOS, NO, NOS, Npn, NRG-3, NT, NTN, OB, OGG1, OPG, OPN, OSM, OX40L, OX40R, p150, p95, PADPr, parathyroid hormone, PA RC, PARP, PBR, PBSF, PCAD, P-cadherin, PCNA, PDGF, PDGF, PDK-1, PECAM, PEM, PF4, PGE, PGF, PGI2, PGJ2, PIN, PLA2, placental alkaline phosphatase (PLAP), PIGF, PLP, PP14, proinsulin, prorelaxin, protein C, PS, PSA, PSCA, prostate-specific membrane antigen (PSMA), PTEN, PTHrp, Ptk, PTN, R51, RANK, RANKL, RANTES, relaxin A-chain, relaxin B-chain, renin, respiratory syncytial virus (RSV) F, RSVFgp, Ret, rheumatoid factor, RLIP76, RPA2, RSK, S100, SCF / KL, SDF-1, SERINE, serum albumin, sFRP-3, Shh, SIGIRR, SK-1, SLAM, SLPI, SMAC, SMDF, SMOH, SOD, SPARC, Stat, STEAP, STEAP-II, TACE, TACI, TAG-72 (tumor-associated glycoprotein-72), TARC, TCA-3, T cell receptor (e.g., T cell receptor alpha / beta), TdT, TECK, TEM1, TEM5, TEM7, TEM8, TERT, testicular PLAP-like alkaline phosphatase, TfR, TGF, TGF-alpha, TGF-beta, TGF-beta pan-specific (Pan Specific), TGF-betaRI (ALK-5), TGF-betaRII, TGF-betaRIIb, TGF-betaRIII, TGF-beta1, TGF-beta2, TGF-beta3, TGF-beta4, TGF-beta5, thrombin, thymic Ck-1, thyroid stimulating hormone, Tie, TIMP, TIQ, tissue factor, TMEFF2, Tmpo, TMPRSS2, TNF, TNF-alpha, TNF-alpha beta, TNF-beta2, TNFc, TNF-RI, TNF-RII, TNFRSF10A (TRAIL R1), Apo-2, DR4, TNFRSF10B (TRAIL R2) DR5, KILLER, TRICK-2A, TRICK-B), TNFRSF10C (TRAIL R3 DcRl, LIT, TRID), TNFRSF10D (TRAIL R4 DcR2, TRUNDD), TNFRSF11A (RANK ODF R, TRANCE R), TNFRSFllB (OPG O THIS、SY1)、SYSYS12 H14) 、SHOCKS H13(SYS)、SHOCKS H130 S)、SHASHS14(SHAS SHAS、SHYS、CHAS S、SY2)、SYSYS16(SYS) 07SYS、SYSYS17(SYSYS)、SYSYS 18(SYS) SHYSY、SYSYS10(SYS3 WHY、WHYWH、WHYWHYWHYWHYWHYWHYWHYWHYWHY 1000000000000000000000000000000000000000000000000000000 H1200、F75806、SHOSH2 6(SYS3)、SYSYS3(SYS THIS SUCH、SHIS S6、SHOSCHS4(SYS)0 SIS33、SHIS1 S) 、WHYS 5(SH40 50)、SWHYS6 DA11、HAR1、DAY5)、DYSCHYS(DYS3 H68、S6)、S6S67(S027)、 FASHION(SHR30)、SHASHES(400) HR137、HARSH、SHRYSY21 DASH) 、SHASH22(DASH) 2 SHY2)、SYSYS23(SYSYS) SHYS1)、SHYSYS25(GY3 FASH3、SHASH、SHASH、SHASH 、SHAKE1)、SHASE100(SHARE). DAY2リガンド、LOY2)、SHAS11(SHAS SHASE RHリガンドDAY 、DAYリガンド)、DAYS12(SIR). DA3リガンド、DAY3リガンド)、DAYDAY13(SYS) LOVE2)、SIGNIFICANCE130 LOVE SY、 LOVE1 LOVE LOVE LOVE LOVE LOVE LOVE LIKEリガンド、THE)、SHASE15(30). SUCH SUCH 18(SUSHリガンドSUBJECTリガンド、CHASE) FASHION、DYS、SYSYS)、SYSYSYS LIKE、SYSYS1)、SYSYS(LOVE THIS、D33) THIS 4(S40リガンドW34、SH1)、SHOSIS5(CHA40リガンドH154、N39、NH10、13 、WATCHES、DAYSYS(THEリガンドApo-1 ligand, APT1 ligand), TNFSF7 (CD27 ligand CD70), TNFSF8 (CD30 ligand CD153), TNFSF9 (4-1BB ligand CD137 ligand), TP-1, t-PA, Tpo, TRAIL, TRAIL R, TRAIL-R1, TRAIL-R2, TRANCE, transfer receptor, TRF, Trk, TROP-2, TSG, TSLP, tumor-associated antigen CA 125, tumor-associated antigens expressing Lewis Y-related carbohydrates, TWEAK, TXB2, Ung, uPAR, uPAR-1, urokinase, VCAM, VCAM-1, VECAD, VE-cadherin, VE-cadherin-2, VEFGR-1 (flt-1), VEGF, VEGFR, VEGFR-3 (flt-4), VEGI, VFM, viral antigens, VLA, VLA-1, VLA-4, VNR integrin, von V Revland factor, WIF-1, WNT1, WNT2, WNT2B / 13, WNT3, WNT3A, WNT4, WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WN T9A, WNT9A, WNT9B, WNT10A, WNT10B, WNT11, WNT16, XCL1, XCL2, XCR1, XCR1, XEDAR, XIAP, XPD, CTLA4 (cytotoxic and at least a second antigen-binding domain that specifically binds to an antigen selected from the group consisting of T lymphocyte antigen-4, PD1 (programmed cell death protein 1), PD-L1 (programmed cell death ligand 1), LAG-3 (lymphocyte activation gene-3), TIM-3 (T cell immunoglobulin and mucin protein-3), a hormone receptor, and a growth factor; (g) The antibody or antigen-binding fragment is selected from the group consisting of BCMA, CTLA4 (cytotoxic T lymphocyte antigen-4), PD1 (programmed cell death protein 1), PD-L1 (programmed cell death ligand 1), LAG-3 (lymphocyte activation ligand 3), and PD-L2 (programmed cell death ligand 2). gene-3), TIM-3, CD20, CD2, CD19, Her2, EGFR, EpCAM, FcyRIIIa (CD16), FcyRIIa (CD32a), FcyRIIb (CD32b), FcyRI (CD64), Toll-like receptors (TLRs), TLR4, TLR9, cytokines, IL-2, IL-5, IL-13, IL-6, IL-17, IL-12, IL-23, TNFa, TGFb, cytokine receptors, IL-2R, chemokines, chemokine receptors, growth factors, VEGF, and HGF; (h) the antibody or antigen-binding fragment is contained in a chimeric antigen receptor (CAR), the CAR optionally comprising at least one transmembrane domain and at least one intracellular domain derived from a T-cell receptor, optionally comprising a CD3ζ subunit, and at least one costimulatory domain; (i) the antibody or antigen-binding fragment comprises scFv2-Fc2 and / or scFv-IgG; (j) the antibody or antigen-binding fragment comprises an IgG constant domain, and / or (k) The anti-CD3 antibody or antigen-binding fragment of any one of claims 1 to 4, wherein the antibody or antigen-binding fragment comprises at least a second antigen-binding domain that specifically binds to an antigen, and the antibody comprises a multispecific format selected from the group consisting of Fab-Fc-scFv, "bottle opener", Mab-scFv, Mab-Fv, dual scFv, central Fv, central scFv, one-arm central scFv, Fab-Fab, Fab-Fv, mAb-Fv, mAb-Fab, DART, BiTE, common light chain-IgG, TandAb, Cross-Mab, SEED, BEAT, TrioMab, and DuetMab.

6. The anti-CD3 antibody and / or antigen-binding fragment of any one of claims 1 to 5, which binds to CD3 or CD3-expressing cells with higher binding affinity at pH 6.0 than at pH 7.

4.

7. An isolated or recombinant nucleic acid sequence encoding the antibody or antigen-binding fragment of any one of claims 1 to 6.

8. 8. An expression vector containing the isolated or recombinant nucleic acid sequence of claim 7.

9. A host cell transfected, transformed or transduced with the nucleic acid sequence of claim 7 or the expression vector of claim 8, wherein the host cell is optionally a mammalian cell or a yeast cell.

10. A pharmaceutical composition comprising the antibody or antigen-binding fragment of any one of claims 1 to 6, or the host cell of claim 9, and a pharmaceutically acceptable carrier and / or excipient.

11. 10. A method of treating a disorder in a mammal in need of such treatment, wherein the disorder comprises a proliferative disorder, an oncological disorder, an immune-oncological disorder, a neurological disorder, a neurodegenerative disorder, or an autoimmune disorder, comprising administering an effective amount of at least one antibody or antigen-binding fragment of any one of claims 1 to 6, or host cells, optionally immune cells, and optionally T cells or NK cells, expressing at least one of the antibodies or antigen-binding fragments of claim 9.

12. 12. The method of claim 11, wherein the method further comprises administering an additional therapeutic agent to the mammal, optionally wherein the mammal is a human.

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