Operated pH-dependent anti-CD3 antibody, and method for producing the same and method for using the same

Abstract

Engineered pH-dependent anti-CD3 binding domains, including multispecific antibodies, with particularly desirable T cell activation and (re)directed target cell killing capabilities and developability profiles, as well as antibodies and / or antigen-binding domains comprising the same, are provided, as are methods for identifying, isolating and producing them, as well as methods for preparing and using them.

Description

Technical Field

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

[0002] Sequence Listing This application is being filed electronically in ASCII format and includes a sequence listing that is hereby incorporated 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] The present invention particularly relates to Heavy engineered pH-dependent anti-surface antigen Cluster of Differentiation 3 (CD3) antibodies that include multispecific antibodies, and functional fragments thereof, as well as methods and reagents for their identification, isolation, preparation, and use.

Background Art

[0004] Cell proliferative disorders, such as cancer, are characterized by the uncontrolled proliferation of cell subsets. 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 cancer cases each year is predicted to reach 23.6 million by 2030. The National Cancer Institute predicts that in 2018, approximately 2 million new cases of cancer will be newly diagnosed in the United States, and more than 600,000 Americans will die of cancer. Therefore, cancer treatment is a major and continuously increasing social burden.

[0005] CD3-targeted bispecific Heavy The idea of using the cytotoxic ability of T cells to kill tumor cells through the use of bispecific antibodies dates back to the mid-1980s. (Staerz et al. Nature 1985 314:628-32). Many of the bispecific HeavyThe specific antibody contains a first binding site specific for CD3 for the recruitment and activation of T cells and a second binding site for a target disease-related antigen such as an antigen produced by tumor cells. CD3 two Heavy By binding to its second target protein expressed on the tumor, the specific antibody induces the CD3 surface receptor on T cells, and as a result, regardless of the peptide / MHC specificity of its T cell receptor, CD3 two Heavy Via cross-linking by the specific antibody, responsive T cells can bind to target-expressing cells. (See, for example, Bassan, 2012, Blood 120:5094-95). CD3 two Heavy By cross-linking T cells and tumor cells using the specific antibody, dramatic regression of advanced malignancies can be induced and in some cases complete remission can also be brought about. For the treatment of malignant blood diseases or solid cancers, targeting CD19, CD20, CD33 and CD123, or EpCAM, HER2, PSMA and CEA respectively, currently, more than 25 various CD3 two Heavy Specific antibodies are in clinical development. (See, for example, Liu et al. Front Immunol 2017 8:38).

[0006] For the treatment and detection of cancer, two Heavy The specific antibody is one Heavy While showing benefits far exceeding those of the specific antibody, two Heavy The widespread commercial application of the specific antibody is inhibited by the lack of an efficient / low-cost production method, two Heavy the lack of stability of the specific polypeptide, and its short half-life in humans. Two Heavy Many methods for producing specific monoclonal antibodies have been developed over the past few decades. However, a number of two Heavy Specific antibody candidates with excellent selectivity and high potency for the target often have issues including the following in downstream development and clinical efficacy: many HeavySpecific binding (or "multispecificity"), off-target binding, non-specific binding, poor expression levels or profiles in eukaryotic host cells such as mammalian host cells or yeast cells, for example, poor stability during storage (e.g., poor / low stability during the "shelf life"), poor solubility (low), poor viscosity (high), poor chemical and physical properties such as a tendency to aggregate, and poor clinical and biophysical profiles such as, for example, poor pharmacokinetic profiles, poor pharmacological profiles, high or poor in vivo clearance rates, short circulation half-lives, etc., some of which contribute to the termination of their development.

[0007] For example, from the perspective of downstream development activities ("post-development antibodies"), such as CIC, SIC, BVP-ELISA, TMA, and other assays, there are specific techniques and assays for evaluating many of the aforementioned developability characteristics for the discovered antibodies. However, such assays often do not fit into the high-throughput format of the initial antibody discovery platform. Furthermore, these trait evaluations often require milligram to gram amounts of protein, and thus, in fact, a practical limitation is imposed on the number of leads that can be realistically considered for development. As a result, the likelihood of program success is reduced. Consequently, vast resources are often spent on attempts to identify lead candidates with poor properties, and there is little available backup at later development stages.

[0008] Monoclonal antibodies and bispecific Heavy Various anti-CD3 antibodies are known in the art, including, for example, monoclonal and bispecific antibody formats. See, for example, U.S. Pat. Nos. 7,262,276, 7,635,472, 7,862,813, 9,587,021, and 10,174,124. However, many of these anti-CD3 antibodies suffer from development problems such as those described above and / or development problems such as the induction of cytokine production, often resulting in harmful cytokine release syndrome (CRS). Bispecific HeavySince the anti-CD3 binding domains of the specific antibodies associate with all T cells, high cytokine-producing CD4 T cell subsets are recruited. Therefore, there is an unmet need for the provision of anti-CD3 antibodies that exhibit a desirable development potential profile and / or CRS risk profile, are safe, and effective, for example, in terms of binding specificity to CD3 expressed on T cells, activation of T cells, redirecting activated T cells to kill target cells, and attenuating the risk of inducing CRS while doing so.

[0009] One way to develop a CD3 binding domain with a desirable CRS risk profile is to engineer a CD3 binding domain 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, for example, Igawa et al., Nature Biotechnology 28:1203-1207 (2010)). Protonation of histidine side chains in the binding interface can change electrostatic interactions and / or induce conformational changes, resulting in differences in the pH-dependence of binding affinity (Gera et al., PLOS ONE 7(11)e48928. doi:10.1371 / 2012). The pH range of human blood is approximately 7.6 - 7.8, whereas tumor cells are recognized to have an extracellular pH of approximately 6.3 - 6.5. The cause of this tumor pH is, at least in part, due to the accumulation of metabolic acids that are not adequately removed due to poor tumor angiogenesis. The engineered pH-dependent CD3 binding domain of the present applicant preferentially binds to CD3 at low pH values, promoting binding and activity within and around the tumor microenvironment. Without being bound by theory, a CD3 binding domain engineered to preferentially bind to CD3 at low pH, such as a pH of approximately 6, for example, may result in selective and sustained cytotoxic activity at or around the tumor site, thereby reducing or eliminating off-target effects, as well as potentially improving half-life and dosage.

SUMMARY OF THE INVENTION

[0010] The present disclosure relates to engineered pH-dependent anti-CD3 antibodies, and antigen-binding fragments thereof, and methods of using the same, which antibodies and antigen-binding fragments 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 present disclosure provides an antibody comprising CDRH3, which is a CD3-binding domain, wherein the CDRH3-binding domain comprises a consensus motif, the consensus motif comprising the sequence AX1DX2YX3HX4FYDV, wherein X1 is R or H, X2 is A or H, X3 is G, H, or P, X4 is Y, H, D, V, E, S, N, L, M, I, G, A, Q, or T, and optionally, at least one of X1, X2, X3, and X4 is replaced with H (SEQ ID NO: 1).

[0015] In some embodiments, the present disclosure provides an antibody comprising CDRH3, which is a CD3-binding domain, wherein the CDRH3-binding domain has a consensus motif represented by the ARDX1YGX2X3X4YDX5 sequence, wherein 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 replaced with H (SEQ ID NO: 2).

[0016] In some embodiments, the present disclosure provides an antibody comprising CDRH3, which is a CD3-binding domain, wherein the CDRH3-binding domain comprises a consensus motif, the consensus motif comprising the ARDAHX1X2YX3X4DX5 sequence, wherein 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 replaced with H (SEQ ID NO: 3).

[0017] In some embodiments, the present disclosure provides an antibody comprising CDRH3, which is a CD3 binding domain, wherein the CDRH3 binding domain comprises a consensus motif, the consensus motif comprises the ARDAX1HRX2FYDV sequence, 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 replaced by H (SEQ ID NO: 4).

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

[0019] In some embodiments, the present disclosure provides an antibody comprising CDRH3, which is a CD3 binding domain, wherein the CDRH3 binding domain comprises a consensus motif, the consensus motif comprises the AX1DAYX2X3X4HX5DV sequence, where X1 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 X1, X2, X3 and X5 is replaced by H (SEQ ID NO: 6).

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

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

[0022] In some embodiments, the present disclosure provides an antibody comprising a CDRH3 that is a CD3 binding domain, the CDRH3 binding domain comprising a consensus motif, the consensus motif comprising the sequence AX1DX2X3X4X5X6X7X8DX9, wherein X1 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 X1, X2, X3, X4, X5, X6, X7, X8 and X9 is H (SEQ ID NO: 58).

[0023] In some embodiments, the present disclosure provides an antibody comprising a CDRH3 that is a CD3 binding domain, the CDRH3 binding domain comprising a consensus motif, the consensus motif comprising the sequence ARDAX1X2X3X4FYDX5, wherein X1 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 X1, X2, X3 and X5 is replaced by H (SEQ ID NO: 593).

[0024] In some embodiments, the present disclosure provides an antibody comprising CDRH3, which is a CD3-binding domain, wherein the CDRH3-binding domain comprises a consensus motif, and the consensus motif comprises 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, X8 is V or H, and optionally, at least one of X1, X2, X3, X4, X5, X7 and X8 is replaced by H (SEQ ID NO: 596).

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

[0026] In some embodiments, the present disclosure provides an antibody comprising CDRH2, which is a CD3-binding domain, wherein the CDRH2-binding domain comprises a consensus motif, and the consensus motif comprises 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 present disclosure provides an antibody comprising CDRH2, which is a CD3-binding domain, wherein the CDRH2-binding domain comprises a consensus motif, and the consensus motif comprises 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 present disclosure provides an antibody comprising CDRH2, which is a CD3 binding domain, wherein the CDRH2 binding domain comprises a consensus motif, and the consensus motif comprises 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 present disclosure provides an antibody comprising CDRH2, which is a CD3 binding domain, wherein the CDRH2 binding domain comprises a consensus motif, and the consensus motif comprises 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 present disclosure provides an antibody comprising a CD3 binding domain, wherein the CDRH2 binding domain comprises a consensus motif, and the consensus motif comprises 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 present disclosure provides an antibody comprising CDRH2, which is a CD3 binding domain, wherein the CDRH2 binding domain comprises a consensus motif, and the consensus motif comprises 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 present disclosure provides an antibody comprising CDRH2, which is a CD3-binding domain, and the CDRH2-binding domain comprises a consensus motif, and the consensus motif comprises the sequence X1INPX2TGX3TKYSQKFQG, wherein 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 present disclosure provides an antibody comprising CDRH2, which is a CD3-binding domain, and the CDRH2-binding domain comprises a consensus motif, and the consensus motif comprises the sequence SIX1AGTGX2TKYSQKFQG, wherein X1 is N or V, and X2 is A or I (SEQ ID NO: 17).

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

[0035] In some embodiments, the present disclosure provides an antibody comprising CDRH2, which is a CD3-binding domain, and the CDRH2-binding domain comprises a consensus motif, and the consensus motif comprises the sequence X1IX2X3GTGX4TDYSQKFQG, wherein 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 present disclosure provides an antibody comprising CDRH2, which is a CD3-binding domain, and the CDRH2-binding domain comprises a consensus motif, and the consensus motif comprises the sequence WIDPX1TGATX2YSQKFQG, wherein X1 is N, H or Y, and X2 is V or K (SEQ ID NO: 20).

[0037] In some embodiments, the present disclosure provides an antibody comprising CDRH2, which is a CD3-binding domain, wherein the CDRH2-binding domain comprises a consensus motif, and the consensus motif comprises 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 present disclosure provides an antibody comprising CDRH2, which is a CD3-binding domain, wherein the CDRH2-binding domain comprises a consensus motif, and the consensus motif comprises the sequence SINAGDANTKYSQKFQG (SEQ ID NO: 22).

[0039] In some embodiments, the present disclosure provides an antibody comprising CDRH2, which is a CD3-binding domain, wherein the CDRH2-binding domain comprises a consensus motif, and the consensus motif comprises 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 present disclosure provides an antibody comprising CDRH2, which is a CD3-binding domain, wherein the CDRH2-binding domain comprises a consensus motif, and the consensus motif comprises the sequence WINAGDAATVYSQKFQG (SEQ ID NO: 24).

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

[0042] In some embodiments, the present disclosure provides an antibody comprising CDRH2, a CD3-binding domain, wherein the CDRH2-binding domain comprises a consensus motif, and the consensus motif comprises 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 present disclosure provides an antibody comprising CDRH1, a CD3-binding domain, 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 present disclosure provides an antibody comprising CDRH1, a CD3-binding domain, wherein the CDRH1-binding domain comprises a consensus motif, and the consensus motif comprises the sequence YTFX1X2X3X4MH, where X1 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 present disclosure provides an antibody comprising CDRH1, which is a CD3-binding domain, wherein the CDRH1-binding domain comprises a consensus motif, and the consensus motif comprises the YTFX1X2X3X4MH sequence, wherein 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 present disclosure provides an antibody comprising CDRH1, which is a CD3-binding domain, wherein the CDRH1-binding domain comprises a consensus motif, and the consensus motif comprises the YTFTSX1X2MH sequence, wherein 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 present disclosure provides an antibody comprising CDRH1, which is a CD3-binding domain, wherein the CDRH1-binding domain comprises a consensus motif, and the consensus motif comprises the YTFX1X2YX3MH sequence, wherein 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 present disclosure provides an antibody comprising CDRH1, which is a CD3-binding domain, wherein the CDRH1-binding domain comprises a consensus motif, and the consensus motif comprises the YTFX1X2YVMH sequence, wherein X1 is I or N, and X2 is K or R (SEQ ID NO: 32).

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

[0050] In some embodiments, the present disclosure provides an antibody comprising CDRH1, which is a CD3-binding domain, wherein the CDRH1-binding domain comprises a consensus motif, and the consensus motif comprises the YTFX1X2YX3MH sequence, 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 present disclosure provides an antibody comprising CDRL3, which is a CD3-binding domain, wherein the CDRL3-binding domain comprises a consensus motif, and the consensus motif comprises the X1X2SX3X4X5RX6 sequence, 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, X6 is T or H, and optionally, at least one of X1, X2, X3, X4, X5 and X6 is replaced by H (SEQ ID NO: 33).

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

[0053] In some embodiments, the present disclosure provides an antibody comprising CDRL3, which is a CD3-binding domain, wherein the CDRL3-binding domain comprises a consensus motif, and the consensus motif comprises the X1QSX2HX3RT sequence, 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 replaced by H (SEQ ID NO: 35).

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

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

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

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

[0058] In some embodiments, the present disclosure provides an antibody comprising CDRL1, a CD3 binding domain, wherein the CDRL1 binding domain comprises a consensus motif, the 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 replaced by H (SEQ ID NO: 38).

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

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

[0061] In some embodiments, the present disclosure provides an antibody comprising CDRL1, a CD3 binding domain, wherein the CDRL1 binding domain comprises 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, X5 is H or Y, and optionally, at least one of X2, X4 and X5 is replaced by H (SEQ ID NO: 42).

[0062] In some embodiments, the present disclosure provides an antibody comprising CDRL1, which is a CD3 binding domain, wherein the CDRL1 binding domain comprises a consensus motif, the 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 replaced by H (SEQ ID NO: 594).

[0063] In some embodiments, the present disclosure provides an antibody comprising CDRL1, which is a CD3 binding domain, wherein the CDRL1 binding domain comprises a consensus motif, the 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 replaced by H (SEQ ID NO: 597).

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

[0065] In some embodiments, the present disclosure provides a CDRH3 binding domain comprising a consensus motif, the consensus motif comprising the AX1DX2X3X4X5X6X7YDX8 sequence, 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, 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 WIDLENANTIYDAKFQG sequence (SEQ ID NO: 9) or the WIDAGTGX1TX2YSQKFQG sequence, 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 FNIKDYYMH sequence (SEQ ID NO: 47) or the YTFX1X2YX3MH sequence, where 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 a consensus motif, the consensus motif comprising the X1QSX2X3X4RT sequence, where X1 is K or H, X2 is Y or H, X3 is S, H, L, V or K, 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 comprising a consensus motif, the consensus motif comprising the WASTRES sequence (SEQ ID NO: 37); a CDRL1 binding domain comprising a consensus motif, the consensus motif comprising the KSSQSLLX1AX2X3X4X5NX6LA sequence, 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 isProvided is an antibody or antigen-binding fragment thereof that is H (SEQ ID NO: 597). In some embodiments, the antibody or antigen-binding fragment is designated as a bispecific 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 present disclosure provides an anti-CD3 antibody or antigen-binding fragment described herein, and the antibody or antigen-binding antibody fragment exhibits a reduced tendency to induce cytokine production to a level capable of inducing cytokine release syndrome while inducing T cell activation or T cell killing.

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

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

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

[0070] In some embodiments, the present disclosure provides an anti-CD3 antibody or antigen-binding fragment described herein, which antibody or antigen-binding fragment may comprise at least a second antigen-binding domain that specifically binds to a tumor target, an immuno-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 blood disease target.

[0071] In some embodiments, the present disclosure provides an anti-CD3 antibody or antigen-binding fragment described herein, which 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-PGFla, 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-1-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 stimulator (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, CIO, 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, welchii toxin, CKb8-1, CLC, CMV, CMVUL, 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-related 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, eotaxinl, 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-alpha1, GFR-alpha2, GFR-alpha3, 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 gp120 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 alpha2, integrin alpha3, integrin alpha4, integrin alpha4 / beta1, integrin, alpha4 / beta7, integrin alpha5 (alphaV), integrin alpha5 / beta1, integrin alpha5 / beta3, integrin alpha6, integrin beta1, integrin beta2, 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, Leftin, 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, lung surfactant, luteinizing hormone, lymphotoxin beta receptor, Mac-1, MAdCAM, MAG, MAP2, MARC, MCAM, MCAM, MCK-2, MCP, M-CSF, MDC, Mer, metalloprotease, 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 duct inhibitory factor, Mug, MuSK, NAIP, NAP, NCAD, N-cadherin, NCA90, NCAM, NCAM, neprilysin, neurotrophin-3, -4, or -6, neurulin, 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, prolactin, 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, TGF-beta RI (ALK-5), TGF-beta RII, TGF-beta Rllb, TGF-beta RIII, 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), TNFRSF1 0C (TRAIL R3 DcRl, LIT, TRID), TNFRSF10D (TRAIL R4 DcR2, TRUNDD), TNFRSF11A (RANK ODF R, TRANCE R), TNFRSF11B (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 TAJ, TRADE), TNFRSF19L (RELT), TNFRSF1A (TNF RI CD120a, p55 - 60), TNFRSF1B (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 (4-1BB 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 Ligand, TL2), TNFSF11 (TRANCE / RANK Ligand ODF, OPG Ligand), TNFSF12 (TWEAK Apo-3 Ligand, DR3 Ligand), TNFSF13 (APRIL TALL2), TNFSF13B (BAFF BLYS, TALL1, THANK, TNFSF20), TNFSF14 (LIGHT HVEM Ligand, LTg), TNFSF15 (TLIA / VEGI), TNFSF18 (GITR Ligand AITR Ligand, TL6), TNFSF1A (TNF-a Conectin, DIF, TNFSF2), TNFSF1B (TNF-b LTa, TNFSF1), TNFSF3 (LTbTNFC, 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, transferrin receptor, TRF, Trk, TROP-2, TSG, TSLP, tumor-associated antigen CA 125, tumor-associated antigen expressing Lewis Y-related sugar, 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 Willebrand 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 receptor, and growth factor.

[0072] In some embodiments, the present disclosure provides an anti-CD3 antibody or antigen-binding fragment described herein, which 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, 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, 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 present disclosure provides an anti-CD3 antibody or antigen-binding fragment described herein, which antibody or antigen-binding antibody fragment may be contained within a chimeric antigen receptor (CAR), and the CAR optionally comprises at least one transmembrane domain, and at least one intracellular domain derived from a T cell receptor, and may optionally comprise a CD3ζ subunit and at least one co-stimulatory domain.

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

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

[0076] In some embodiments, the present disclosure provides an anti-CD3 antibody or antigen-binding fragment described herein, which antibody or antigen-binding antibody fragment may comprise at least a second antigen-binding domain that specifically binds to an antigen, in which case the antibody comprises a multispecific format selected from the group consisting of: Fab-Fc-scFv, "plug-and-play", 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 an isolated nucleic acid sequence or recombinant nucleic acid sequence encoding an anti-CD3 antibody or antigen-binding fragment described herein.

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

[0079] In some embodiments, the present disclosure provides a host cell 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 nucleic acid sequence or recombinant nucleic acid sequence encoding an anti-CD3 antibody or antigen-binding fragment described herein, which 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 a method of treating a disorder in a mammal in need of such treatment, the disorder may include a proliferative disorder, a neoplastic disorder, an immuno-oncological disorder, a neurological disorder, a neurodegenerative disorder, or an autoimmune disorder, and the method may include administering an effective amount of at least one antibody or antigen-binding fragment described herein, or a host cell expressing one of the antibodies or antigen-binding fragments described herein, optionally an immune cell, and further optionally a T cell or an NK cell. In some embodiments, the method may further include administering an additional therapeutic agent to the mammal, and optionally in this case the mammal is a human.

[0082] In other embodiments, the present disclosure provides an anti-CD3 antibody or an antigen-binding fragment thereof comprising one or more of CDRL1, CDRL2, and CDRL3. Such an antibody may further include CDRH1, CDRH2, and CDRH3 in some embodiments. The present disclosure relates to, for example, the following. [1] An anti-cluster of differentiation 3 (CD3) antibody and / or antigen-binding fragment thereof, (a) i. AX 1 DX 2 X 3 X 4 X 5 X 6 X 7 X 8 DX 9 wherein X 1 is R or H, X 2 is A, H, M or Q, X 3 is Y, H, S, G, A, T, V or R, X 4 is G, H, P, E or R, X 5 is H or R, X 6 is Y, N, F, H, D, E, S, L, M, I, G, A, Q or T, X 7 is F or H, X 8 is Y or H, 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 at least one of which is H (SEQ ID NO: 58), ii. ARDX 1 X 2 X 3 X 4 YFYDX 5 wherein X 1 is H or A, X 2 is T, Y or H, X 3 is G or H, X 4 is H, R, V or I, X 5 is V or H, and optionally, X 1 、X 2 、X 3 、X 4 and X 5 at least one of which is H (SEQ ID NO: 43), iii. AX 1 DX 2 YX 3 HX 4 FYDV wherein X 1 is R or H, X 2 is A or H, X 3 is G, H or P, 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 at least one of which is H (SEQ ID NO: 1), iv. ARDX 1 YGX 2 X 3 X 4 YDX 5 wherein X 1 is A or H, X 2 is R or H, X 3 is H or Y, X 4 is F or H, X 5 is H or V, and optionally, X 1 、X 2 、X 3 、X 4 , and X 5 at least one of which is H (SEQ ID NO: 2), v. ARDAHX 1 X 2 YX 3 X 4 DX 5 wherein X 1 is G, E or R, X 2 is R or H, X 3 is F or H, X 4 is Y or H, X 5 is V or H, and optionally, X 1 、X 2 、X 3 、X 4 and X 5 at least one of which is H (SEQ ID NO: 3), vi. ARDAX 1 HRX 2 FYDV wherein X 1 is H, Y, S, G, A, T, V or R, X 2 is Y or H, and optionally, X 1 and X 2 at least one of which is H (SEQ ID NO: 4), vii. ARDX 1 YHRYFYDX 2 wherein X 1 is H or A, X 2 is H, V or M, and optionally, X 1 and X 2 at least one of which is H (SEQ ID NO: 5), viii. AX 1 DAYX 2 X 3 X 4 HX 5 DV wherein X 1 is R or H, X 2 is G or H, X 3 is H or R, X 4 is N, F or Y, X 5 is Y or H, and optionally, X 1 、X 2 、X 3 、X 4 and X 5 at least one of which is H (SEQ ID NO: 6), ix. ARDX 1 X 2 GRYFYDV, X 1 is M, Q or H, X 2 is R or H, and optionally, X 1 and X 2 at least one of which is H (SEQ ID NO: 7), x. ARDX1X2X3RYFYDX4, X 1 is H or A, X 2 is T, Y or H, X 3 is G or H, X 4 is V or H, and optionally, X 1 、X 2 、X 3 , and X 4 at least one of which is H (SEQ ID NO: 8), xi. ARDAX 1 X 2 X 3 X 4 FYDX 5 , X 1 is T, H or Y, X 2 is G or H, X 3 is H or R, X 4 is V or Y, X 5 is V or H, and optionally, X 1 、X 2 、X 3 and X 5 at least one of which is H (SEQ ID NO: 593), and xii. AX 1 DX 2 X 3 X 4X 5 X 6 X 7 YDX 8 , X 1 is R or H, X 2 is H or A, X 3 is H or Y, X 4 is H, G or P, X 5 is R or H, X 6 is Y, I or V, X 7 is F or H, X 8 is V or H, and optionally, X 1 、X 2 、X 3 、X 4 、X 5 、X 7 and X 8 at least one of which is H (SEQ ID NO: 596) selected from the group consisting of V H chain CDR3 (CDRH3) - containing variable heavy (V H ) chain polypeptide, (b) i. X 1 IX 2 X 3 X 4 X 5 X 6 X 7 TX 8 YSQKFQG, X 1 is W, S, Y, F, G or D, X 2 is N, T, D, V or H, X 3 is A, P or S, X 4 is G, A, S, N, D, L, V, H, Q, T, I or Y, X 5 is D or T, X 6 is A or G, X 7 is A, N, T, S, L, D, F, Y or E, 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 at least one of which is H (SEQ ID NO: 59), ii. WIDLENANTIYDAKFQG (SEQ ID NO: 9), iii. X 1 INPX 2 TGX 3 TX 4 YSQKFQG, X 1 is W or Y, X 2 is A, S, D, G, N, L, V, H or Q, X 3 is A, T or S, 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 3is A, T or L, and X 4 is one of 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 one of 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, and X 3 is one of 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 one of 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 one of I or V (SEQ ID NO: 15), ix. X 1 INPX 2 TGX 3 TKYSQKFQG, where X 1 is W or Y, and X 2 is D, I or Y, and X 3 is one of D, Y or E (SEQ ID NO: 16), x. SIX 1 AGTGX 2 TKYSQKFQG, where X 1 is N or V, and X 2 is one of A or I (SEQ ID NO: 17), xi. SINAGTGX 1 TX 2 YSQKFQG, where X 1 is F or N, and X 2 is one of 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 one of 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 one of V or K (SEQ ID NO: 20), xiv. WIX 1 PX 2 TGNTKYSQKFQG, where X 1 is D or N, and X 2is one of L, I or V (SEQ ID NO: 21), xv. SINAGDANTKYSQKFQG (SEQ ID NO: 22), and 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 one of 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 those which are T or K (SEQ ID NO: 595) H Variable heavy (V H ) chain polypeptide comprising a locked CDR2 (CDRH2), (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, wherein X 1 is A, K, D, Q, E, N, T, L, Y, S, P, G, H or V, X 2 is T, S or A, X 3 is Y or I, 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, wherein X 1 is T, D, A, N or V, X 2 is D, E, G or Q, X 3 is Y or D, X 4 is D, A, E, N, S, Y or V (SEQ ID NO: 29), vi. YTFTSX 1 X 2 MH, wherein X 1 is A, D or T, X 2 is D, F, A, M, V or Y (SEQ ID NO: 30), vii. YTFX 1 X 2 YX 3 MH, wherein X 1 is N or T, X 2 is Q or N, X 3 is S, T or A (SEQ ID NO: 31), and viii. YTFX 1 X 2 YVMH, wherein X 1 is I or N, 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, wherein X 1 is E, S or T, X 2 is S or D, X 3 is A or D (SEQ ID NO: 31) selected from the group consisting of V H chain CDR1 (CDRH1) comprising variable heavy (V H ) chain polypeptide, (d) i. X 1 X 2 SX 3 X 4 X 5 RX 6 , wherein X 1 is H, K or G, X 2 is Q or H, X 3 is Y or H, X 4 is S, H, D, T, V, M or L, X 5 is R or H, X 6 is T or H, and optionally, X 1 、X 2 、X 3 、X 4 、X 5 and X 6 at least one of which is H (SEQ ID NO: 33), ii. KQSYX 1 X 2 RT, wherein X 1 is H, V, K, W, R, L, G, Y or Q, X 2 is H, L, E, W, G, M, P, T, Q or V, and optionally, X 1 and X 2 at least one of which is H (SEQ ID NO: 34), iii. X 1 QSX 2 HX 3 RT, wherein X 1 is K or H, X 2 is H, Y, M, S, L, E, G or W, X 3 is R or K, and optionally, X 1 and X 2 at least one of which is H (SEQ ID NO: 35), iv. KQSX 1 X 2 X 3 RT, wherein X 1 is Y or H, X 2 is T, S, V or K, X 3 is R or H, and optionally, X 1 and X 3 at least one of which is H (SEQ ID NO: 36), v.KQSX 1 X 2 X 3 RT, where X 1 is H or Y, X 2 is T, S or Q, X 3 is R or H, and optionally, X 1 and X 3 at least one of which is H (SEQ ID NO: 36), and vi.X1 QSX 2 X 3 X 4 RT, where X 1 is K or H, X 2 is Y or H, X 3 is S, H, L, V or K, X 4 is H, R or E, and optionally, X 1 、X 2 、X 3 and X 4 at least one of which is H (SEQ ID NO: 598) selected from the group consisting of V L chain CDR3 (CDRL3) containing variable light (V L ) chain polypeptide, (e) variable light (V L ) chain polypeptide containing chain CDR2 (CDRL2) of WASTRES (SEQ ID NO: 37), and / or L i.KSSQSLLX (f) GX 1 X 2 X 3 X 4 NX 5 LA, where X 6 is N or H, X 1 is A, R or T, X 2 is R or H, X 3 is T, P or E, X 4 is H or K, X 5 is H or Y, and optionally, X 6 and X 1 、X 3 、X 5 at least one of which is H (SEQ ID NO: 38), 6 ii.KSSQSLLX AX 1 THX 2 NX 3 LA, where X 4 is N or H, X 1 is R or H, X 2 is K or H, X 3 is Y or H, and optionally, X 4 and X 1 、X 2 、X 3 at least one of which is H (SEQ ID NO: 39), 4 iii.KSSQSLLNASTAKNYLA (SEQ ID NO: 40), iv.KSSQSLLNARTRTNYLA (SEQ ID NO: 41), v.KSSQSLLNX GX 1 X 2 X 3 NX 4 LA, where X 5 is S or A, X 1 is R or H, X 2 is E or T, X 3 is H or K, X 4 is H or Y, and optionally, X 5 and X 2 、X 4at least one of which is H (SEQ ID NO: 42), 5 vi.KSSQSLLNX TGX 1 X 2 NYLA, where X 3 is A or S, X 1 is R or H, X 2 is H or K, and optionally, X 3 and X 2 at least one of which is H (SEQ ID NO: 594), and 3 vii.KSSQSLLX AX 1 NX 2 X 3 X 4 X 5 LA, where X 6 is N or H, X 1 is R or H, X 2 is T or E, X 3 is G or H, X 4 is H or K, X 5 is H or Y, and optionally, X 6 ​ 1 、X 2 、X 4 、X 5 and X 6 V selected from the group consisting of those in which at least one of is H (SEQ ID NO: 597) L chain CDR1 (CDRL1) containing a variable light (V L ) chain polypeptide, an anti-CD3 antibody and / or antigen-binding fragment comprising the same. [2] (a) The amino acid sequence ARDX 1 X 2 X 3 X 4 YFYDX 5 wherein X 1 is H or A, X 2 is T, Y or H, X 3 is G or H, X 4 is H, R, V or I, X 5 is V or H, and optionally, X 1 、X 2 、X 3 、X 4 and X 5 CDRH3 containing at least one of which is H (SEQ ID NO: 43), (b) The amino acid sequence KQSX 1 X 2 X 3 RT wherein X 1 is Y or H, X 2 is T, S, V or K, X 3 is R or H, and optionally, X 1 and X 3 CDRL3 containing at least one of which is H (SEQ ID NO: 36), and / or (c) The amino acid sequence KSSQSLLNX 1 X 2 X 3 GX 4 NX 5 LA wherein X 1 is S or A, X 2 is R or H, X3 is E or T, X 4 is H or K, X 5 is H or Y, and optionally, X 2 、X 4 and X 5 CDRL1 containing at least one of which is H (SEQ ID NO: 42), comprising the anti-CD3 antibody and / or antigen-binding fragment according to item 1. [3] (a) The amino acid sequence ARDAX 1 X 2 X 3 X 4 FYDX 5 wherein X 1 is T, H or Y, X 2 is G or H, X 3 is H or R, X 4 is V or Y, X 5 is V or H, and optionally, X 1 、X 2 、X 3 and X 5 CDRH3 containing at least one of which is H (SEQ ID NO: 593), (b) CDRH2 containing the amino acid sequence WIDLENANTIYDAKFQG (SEQ ID NO: 9), (c) CDRH1 containing the amino acid sequence FNIKDYYMH (SEQ ID NO: 47), (d) The amino acid sequence KQSX 1 X 2 X 3 RT wherein X 1 is H or Y, X 2 is T, S or Q, X 3 is R or H, and optionally, X 1 and X 3 CDRL3 containing at least one of which is H (SEQ ID NO: 36), (e) CDRL2 containing the amino acid sequence WASTRES (SEQ ID NO: 37), and / or (f) The amino acid sequence KSSQSLLNX 1 X 2 TGX 3 NYLA wherein X 1 is A or S, X 2 is R or H, X 3 is H or K, and optionally, X 2 and X 3 The anti-CD3 antibody or antigen-binding fragment according to claim 1, comprising CDRL1 that includes at least one of them being H (SEQ ID NO: 594). [4] (a) The amino acid sequence AX 1 DX 2 X 3 X 4 X 5 X 6 X 7 YDX 8 wherein X 1 is R or H, X 2 is H or A, X 3 is H or Y, X 4 is H, G or P, X 5 is R or H, X 6 is Y, I or V, X 7 is F or H, X 8 is V or H, and optionally, at least one of X 1 、X 2 、X 3、X 4 、X 5 、X 7 and X 8 is H (SEQ ID NO: 596)-containing CDRH3, (b) i. WIDLENANTIYDAKFQG (SEQ ID NO: 9), and ii. WIDAGTGX 1 TX 2 YSQKFQG wherein X 1 is L, F, N or A, X 2 is T or K, and the CDRH2 selected from the group consisting of the amino acid sequences (SEQ ID NO: 595), (c) i. FNIKDYYMH (SEQ ID NO: 47), and ii. YTFX 1 X 2 YX 3 MH wherein X 1 is E, S or T, X 2 is S or D, X 3 is A or D, and the CDRH1 selected from the group consisting of the amino acid sequences (SEQ ID NO: 31), (d) The amino acid sequence X 1 QSX 2 X 3 X 4 RT wherein X 1 is K or H, X 2 is Y or H, X 3 is S, H, L, V or K, X 4 is H, R or E, and optionally, at least one of X 1 、X 2 、X 3 and X 4 is H (SEQ ID NO: 598)-containing CDRL3, (e) CDRL2 containing the amino acid sequence WASTRES (SEQ ID NO: 37), and / or (f) The amino acid sequence KSSQSLLX 1 AX 2 X 3 X 4 X 5 NX 6 LA wherein X 1 is N or H, X 2 is R or H, X 3 is T or E, X 4 is G or H, X 5 is H or K, X 6 is H or Y, and optionally, at least one of X 1 、X 2 、X 4 、X 5 and X 6 is H (SEQ ID NO: 597)-containing CDRL1, the anti-CD3 antibody or antigen-binding fragment according to claim 1. [5] (a) The antibody or antigen-binding fragment exhibits a reduced tendency to induce cytokine production to a level capable of inducing cytokine release syndrome while inducing T cell activation or T cell killing, (b) The antibody or antigen-binding fragment includes a multispecific antibody, (c) The antibody or antigen-binding fragment includes a bispecific antibody, (d) The antibody or antigen-binding fragment includes an scFv, (e) The antibody or antigen-binding fragment thereof comprises at least a second antigen-binding domain that specifically binds to a tumor target, an immuno-oncology target, a neurodegenerative disease target, an autoimmune disorder target, an infectious disease target, a metabolic disease target, a cognitive impairment target, a blood-brain barrier target, or a blood disease target. (f) The antibody or antigen-binding fragment is 17-IA, 4-1BB, 4Dc, 6-keto-PGFla, 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-1-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 stimulator (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, CIO, 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-l, 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-related 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, eotaxinl, 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, GFRa-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:Hemopoietic 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:High molecular weight melanoma-associated antigen), 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 alpha2, integrin alpha3, integrin alpha4, integrin alpha4 / beta1, integrin, alpha4 / beta7, integrin alpha5 (alphaV), integrin alpha5 / beta1, integrin alpha5 / beta3, integrin alpha6, integrin beta1, integrin beta2, 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 - 1 bpl, 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, luteinizing hormone, lymphotoxin beta receptor, Mac - 1, MAdCAM, MAG, MAP2, MARC, MCAM, MCAM, MCK - 2, MCP, M - CSF, MDC, Mer, metalloprotease, MGDF receptor Vesicle, 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, 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, Prolactin, 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, RSV Fgp, 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 (PanSpecific), TGF-beta RI (ALK-5), TGF-beta RII, TGF-beta RIIb, TGF-beta RIII, TGF-beta 1, 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 R1 Apo-2, DR4), TNFRSF10B (TRAIL R2 DR5, KILLER, TRICK-2A, TRICK-B), TNFRSF10C (TRAIL R3 DcR1, LIT, TRID), TNFRSF10D (TRAIL R4 DcR2, TRUNDD), TNFRSF11A (RANK ODF R, TRANCE R), TNFRSF11B (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 TAJ, TRADE), TNFRSF19L (RELT), TNFRSF1A (TNF RI CD120a, p55-60), TNFRSF1B (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 (4-1BB CD137, ILA), TNFRSF21 (DR6), TNFRSF22 (DcTRAIL R2 TNFRH2), TNFRST23 (DcTRAIL R1 TNFRH1), TNFRSF25 (DR3Apo-3, LARD, TR-3, TRAMP, WSL-1), TNFSF10 (TRAIL Apo-2 ligand, TL2), TNFSF11 (TRANCE / RANK ligand ODF, OPG ligand), TNFSF12 (TWEAK Apo-3 ligand, DR3 ligand), TNFSF13 (APRIL TALL2), TNFSF13B (BAFF BLYS, TALL1, THANK, TNFSF20), TNFSF14 (LIGHT HVEM ligand, LTg), TNFSF15 (TLIA / VEGI), TNFSF18 (GITR ligand AITR ligand, TL6), TNFSF1A (TNF-a Conectin, DIF, TNFSF2), TNFSF1B (TNF-b LTa, TNFSF1), TNFSF3 (LTb TNFC, 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 (transferring receptor), TRF, Trk, TROP-2, TSG, TSLP, tumor-associated antigen CAAt least a second antigen-binding domain that specifically binds to an antigen selected from the group consisting of 125, tumor-associated antigens expressing Lewis Y-related sugars, 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 Willebrand 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. (g) The antibody or antigen-binding fragment thereof comprises at least a second antigen-binding domain that specifically binds to an antigen 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 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 thereof is contained within 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, optionally a CD3ζ subunit, and at least one co-stimulatory domain. (i) The antibody or antigen-binding fragment thereof comprises scFv2-Fc2 and / or scFv-IgG. (j) The antibody or antigen-binding fragment thereof comprises an IgG constant domain and / or (k) The antibody or antigen-binding fragment thereof comprises at least a second antigen-binding domain that specifically binds to an antigen, and the antibody has a multispecific format selected from the group consisting of Fab-Fc-scFv, "plug-and-play", 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. The anti-CD3 antibody or antigen-binding fragment according to any one of items 1 to 4. [6] The anti-CD3 antibody and / or antigen-binding fragment according to any one of items 1 to 5, which binds to CD3 or CD3-expressing cells with high binding affinity at pH 6.0 rather than pH 7.4. [7] An isolated nucleic acid sequence or recombinant nucleic acid sequence encoding the antibody or antigen-binding fragment according to any one of items 1 to 6. [8] An expression vector containing the isolated nucleic acid sequence or recombinant nucleic acid sequence according to item 7. [9] A host cell transfected, transformed, or transduced with the nucleic acid sequence according to item 7, or the expression vector according to item 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 according to any one of items 1 to 6, or the host cell according to item 9, and a pharmaceutically acceptable carrier and / or excipient.

[11] A method for treating a disorder in a mammal in need of such treatment, wherein the disorder includes a proliferative disorder, a neoplastic disorder, an immuno-oncological disorder, a neurological disorder, a neurodegenerative disorder, or an autoimmune disorder, and the method comprises administering an effective amount of at least one antibody or antigen-binding fragment according to any one of items 1 to 6, or the host cell expressing at least one of the antibodies or antigen-binding fragments according to item 9, optionally an immune cell, and further optionally a T cell or an NK cell.

[12] The method according to item 11, further comprising administering an additional therapeutic agent to the mammal, and optionally the mammal is a human.

Brief Description of the Drawings

[0083]

Figure 1

[0084]

Figure 2

[0085]

Figure 3

[0086]

Figure 4

[0087]

Figure 5

[0088]

Figure 6

Mode for Carrying Out the Invention

[0089] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. As used herein, the term "about," when used in connection with a specific recited numerical value, means that the value can vary by up to 1% from the recited value. For example, as used herein, the expression "about 100" includes 99 and 101, as well as all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0090] It should be understood that the aspects and embodiments of the present disclosure described herein include those that "comprise", "consist of", and "consist essentially of" the 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 derived from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), and includes, unless otherwise indicated, for example, the CD3ε chain, CD3γ chain, CD3α chain, and CD3β chain. The term encompasses "full-length", unprocessed CD3 (e.g., unprocessed or unmodified CD3ε or CD3γ) as well as any form of CD3 resulting from processing in cells. The term also encompasses native variants of CD3, such as splice variants or allelic variants. Examples of CD3 include, for example, the human CD3ε protein (NCBI reference sequence number NP _ 000724), which is 207 amino acids in length. And the human CD3γ protein (NCBI reference sequence number NP _ 000064) is also included, which is 182 amino acids in length. The term also refers to the human or cynomolgus monkey CD3ε protein, which are SEQ ID NOs: 591 and 592, respectively (Table 4). "CD3εN27" and "CD3εN13" refer to the 27 amino acids at the N-terminus and the 13 amino acids at the N-terminus of CD3, respectively, and optionally contain chemical modifications or linkages applied thereto.

[0093] The term "anti-CD3 antibody" refers to an antibody or an antigen-binding fragment thereof that has the ability to bind to CD3, such as CD3ε and / or CD3γ, for example, human CD3ε and / or CD3γ, and is accompanied by sufficient affinity and / or specificity such that the antibody is useful as a diagnostic agent and / or therapeutic agent in CD3 targeting. In some embodiments, the anti-CD3 antibody has a dissociation constant (K -9 d) of about 100×10 -9 −9 M or less, about 50×10 -9 −9 M or less, about 25×10 -9 −9 M or less, about 20×10 -9 −9 M or less, or about 10×10 D −9 M or less for binding to CD3. In some embodiments, the anti-CD3 antibody has a dissociation constant (K -9 d) of about 5×10 D −9 M or less for binding to CD3. In some embodiments, the anti-CD3 antibody has a dissociation constant (K -9 d) of about 2.5×10 D −9 M or less for binding to CD3. In some embodiments, the anti-CD3 antibody has a dissociation constant (K -10 d) of about 1×10 D −9 M or less for binding to CD3. In some embodiments, K D d is measured by surface plasmon resonance, such as BIACORE, by biolayer interferometry using, for example, a FORTEBIO Octet HTX instrument (Pall Life Sciences), or by solution-affinity ELISA. In some embodiments, KD is measured using the scFv fragment of the anti-CD3 antibody. In some embodiments, the monovalent KD is measured. In some embodiments, the anti-CD3 antibody binds to an epitope of CD3 that is conserved among CD3s from various species, such as cross-reactive species of human and cynomolgus monkey.

[0094] The term "engineered pH-dependence" refers to an antibody having a modified amino acid sequence that enables 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 bind in a pH-dependent manner. pH-dependent binding refers to a preference of an antibody to bind an antigen at a given pH (or given pH range) as compared to different pHs (or pH ranges). In one embodiment, a pH-dependent antibody preferentially or selectively binds an antigen at a pH of approximately 6 as compared to a pH of approximately 7. The antibody sequence may be modified, for example, by substitution with one or more ionizable amino acid residues such as histidine, lysine, arginine, aspartic acid, and glutamic acid. The 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 an excessive and uncontrolled release of pro-inflammatory cytokines by cells within the immune system (see, e.g., 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 series of cytokines to levels and extents that cause harmful biological / physiological effects of varying degrees and severities. Such effects include, for example, acute inflammation characterized by erythema (redness), swelling or edema, fever (flushing), pain (ache), and loss of function. When localized to the skin or other tissues, the biological / physiological effects include an increase in blood flow, an increase in local temperature allowing vascular leukocytes and plasma proteins to reach extravascular sites of injury, as well as the development of pain, tissue edema and extravascular pressure, and a decrease in tissue perfusion. Other biological / physiological effects include organ and systemic dysfunction, such as heart failure, adult respiratory distress syndrome, neurotoxicity, renal failure and / or liver failure, and disseminated intravascular coagulation syndrome. 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 are involved as indicators and / or causes of CRS, or the tendency to induce CRS upon T cell stimulation.

[0096] In certain embodiments, the anti-CD3 antibodies and / or antigen-binding fragments thereof described herein are degraded 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 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, and polyclonal antibodies. Heavy Specific antibodies (e.g. Heavy Antibody-specific antibodies, and / or antibody fragments (preferably fragments exhibiting the desired antigen-binding activity) are included.

[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 the 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] Many Heavy For specific antibodies, such antibodies contain at least two different antigen-binding domains, which recognize and specifically bind to at least two different antigens. Heavy Regarding specific antibodies, such antibodies contain two different antigen-binding domains, which recognize and specifically bind at least two different antigens.

[0100] "Different antigens" may refer to different and / or distinct proteins, polypeptides, or molecules, and may also refer to different and / or distinct epitopes. Such epitopes may be contained within one 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 a paratope. An antigen may have multiple epitopes. Thus, different antibodies may bind to different regions on 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. Additionally, it refers to the region of an antigen that binds to an antibody. Epitopes may be defined structurally or functionally. Functional epitopes are generally a subset of structural epitopes and have residues that directly contribute to the affinity of the interaction. Epitopes may also be conformational, i.e., composed of non-linear amino acids. In certain embodiments, an epitope may include determinants that are chemically active surface populations of molecules such as, for example, amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups. In certain embodiments, an epitope may have specific three-dimensional structural characteristics and / or specific charge characteristics.

[0102] In some examples, an antibody comprises four polypeptide chains, i.e., 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, and some of these may be further divided into subclasses (isotypes) such as, for example, IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy-chain constant domains corresponding to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0103] In other examples, the antibody may alternatively comprise its multimeric form (e.g., IgM) or its antigen-binding fragment. Each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region, and the heavy chain constant region is 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 named framework regions (FR) and hypervariable regions named complementarity-determining regions (CDR) disposed 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 FR of the antibody (or its antigen-binding fragment) may be identical to the sequence of the human germline, or may be natural, or may be artificially modified. The consensus sequence of amino acids may be defined based on the result of aligning two or more CDRs. Thus, the CDRs of the heavy chain are designated "CDRH1", "CDRH2", and "CDRH3", respectively, and the CDRs of the light chain are designated "CDRL1", "CDRL2", and "CDRL3".

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

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

[0106] The antigen-binding fragment of an antibody includes any natural, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex, and includes antibody fragments. Exemplary antigen-binding fragments include, but are not limited to, Fv, Fab, Fab’, Fab’-SH, F(ab’)2, diabody, linear antibody, single-chain antibody molecule (e.g., scFv, or only the VH domain or VL domain), and multispecific antibodies formed from antibody fragments. In some embodiments, the antigen-binding fragment of the anti-CD3 antibody described herein is an scFv.

[0107] Similar to a complete antibody molecule, an antigen-binding fragment is Heavy either monospecific or multi Heavy specific (e.g., bispecific). Heavy The multispecific antigen-binding fragment of an antibody may contain at least two different variable domains, in which case each variable domain can specifically bind to a distinct antigen or can specifically bind to different epitopes on the same antigen. In the case of the antigen-binding fragment of the anti-CD3 antibody described herein, various multi Heavy specific antibody formats can be used. Multi Heavy specific formats and bispecific Heavy formats are described herein. Heavy Non-limiting examples of specific formats include, for example, Fab-Fc-scFv (plug-and-play 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 (SFFIMED), Cross-Mab (ROCHE), SEED (EMD SERONO), BEAT (GLENMARK), TrioMab (TRION PHARMA / FRESENIUS BIOTECH), DuetMab (MEDIMMUNE), etc., which are disclosed, 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; WO2016 / 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 fragment described herein is multi Heavy specificity (e.g., two Heavy specificity) antibodies and includes one or more variable domains.

[0108] In certain embodiments, the anti-CD3 antibodies and / or antigen-binding fragments thereof described herein are multi Heavy specific antibodies, particularly bispecific antibodies having binding specificity for a second antigen, Heavy contained within the bispecific antibodies. Such a second antigen may be a completely different target from the first target, or may be a different epitope present on the same target. In some embodiments, the binding specificity is binding specificity 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 biomolecule (e.g., a cell surface antigen, e.g., a tumor antigen).

[0109] Non-limiting examples of a second antigen to which a bispecific antibody comprising an anti-CD3 antibody and / or an antigen-binding fragment thereof described herein is directed include targets selected from the group consisting of: 17-IA, 4-1BB, 4Dc, 6-keto-PGFla, 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-1-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 stimulator (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, 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, 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, welchii toxin, CKb8-1, CLC, CMV, CMVUL, 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, Disintegrin, 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, 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, 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-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 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 gp120 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 alpha2, integrin alpha3, integrin alpha4, integrin alpha4 / beta1, integrin, alpha4 / beta7, integrin alpha5 (alphaV), integrin alpha5 / beta1, integrin alpha5 / beta3, integrin alpha6, integrin beta1, integrin beta2, 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, Leftie, 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, luteinizing hormone, lymphotoxin beta receptor, Mac-1, MAdCAM, MAG, MAP2, MARC, MCAM, MCAM, MCK-2, MCP, M-CSF, MDC, Mer, metalloprotease, 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 duct inhibitory factor, Mug, MuSK, NAIP, NAP, NCAD, N-cadherin, NCA90, NCAM, NCAM, neprilysin, neurotrophin-3, -4, or -6, neurulin, 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, prolactin, protein C, PS, PSA, PSCA, prostate specific membrane antigen (PSMA), PTEN, PTHrp, Ptk, PTN, R51, RANK, RANKL, RANTES, 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, TGF-beta RI (ALK-5), TGF-beta RII, TGF-beta Rllb, TGF-beta RIII, TGF-beta 1, 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), TNFRSF10C (TRAIL R3 DcRl, LIT , TRID), TNFRSF10D (TRAIL R4 DcR2, TRUNDD), TNFRSF11A (RANK ODF R, TRANCE R), TNFRSF11B (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 TAJ, TRADE), TNFRSF19L (RELT), TNFRSF1A (TNF RI CD120a, p55 - 60), TNFRSF1B (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 - 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 Ligand, TL2), TNFSF11 (TRANCE / RANK Ligand ODF, OPG Ligand), TNFSF12 (TWEAK Apo - 3 Ligand, DR3 Ligand), TNFSF13 (APRIL TALL2), TNFSF13B (BAFF BLYS, TALL1, THANK, TNFSF20), TNFSF14 (LIGHT HVEM Ligand, LTg), TNFSF15 (TL1A / VEGI), TNFSF18 (GITR Ligand AITR Ligand, TL6), TNFSF1A (TNF - a Conectin, DIF, TNFSF2), TNFSF1B (TNF - b LTa, TNFSF1), TNFSF3 (LTb TNFC, p33), TNFSF4 (OX40 Ligandgp34, 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, tumor-associated antigen expressing Lewis Y-related sugar, 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 Willebrand 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 receptor, and growth factor.

[0110] The multispecific substances comprising the anti-CD3 antibodies and antigen-binding fragments disclosed herein may be prepared according to various techniques including, but not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs having different specificities (see Milstein and Cuello, Nature 305:537 (1983), WO93 / 08829, and Traunecker et al., EMBO J. 10:3655 (1991)), "knob-in-hole" engineering (see, e.g., U.S. Patent No. 5,731,168), immunoglobulin crossover (also known as Fab domain exchange or CrossMab format) technology (see, e.g., WO2009 / 080253; Schaefer et al., Proc. Natl. Acad. Sci. USA, 108:11187-11192 (2011)), engineering electrostatic steering effects for the production of antibody Fc heterodimer molecules (WO2009 / 089004A1), crosslinking of two or more antibodies or fragments (see, e.g., U.S. Patent No. 4,676,980, and Brennan et al., Science, 229:81 (1985)), leucine zipper (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 such as those described in, e.g., Tutt et al. J. Immunol 147:60 (1991).

[0111] The present disclosure also contemplates modifications of the anti-CD3 antibodies disclosed herein, such modifications including substitutions, insertions, and / or deletions of one or more amino acids in the FR and / or CDR regions of the heavy chain variable domain and the light chain variable domain. Upon acquisition, such induced antibodies and / or antigen-binding fragments may be verified with respect to one or more desirable properties such as improved binding specificity, increased binding affinity, improved developability, and the like.

[0112] In some embodiments, the anti-CD3 antibody and / or its antigen-binding fragment 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 antibody and / or its antigen-binding fragment has amino acid sequence identity to the corresponding sequences of the anti-CD3 antibodies disclosed in Table 1 (Ab1-Ab258) 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 all percentages in between. In some embodiments, the percent identity is measured by any known algorithm for sequence identity such as, for example, FASTA, BLAST, or GAP.

[0113] In some embodiments, non-identical residue positions have different conservative amino acid substitutions. A "conservative amino acid substitution" is a substitution in which an amino acid residue is replaced by 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 the protein. When two or more amino acid sequences differ from each other by conservative substitutions, the percent similarity or degree may be adjusted upward to account for the conservative nature of the substitutions. 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 having side chains with 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, the group of conservative amino acid substitutions is valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine. Alternatively, in some embodiments, conservative substitutions include any change having a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256:1443-45. In some embodiments, "moderately conservative" substitutions include any change having 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. In the scientific literature, antibodies have been reported in which one or two CDRs are missing 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 relevant antigens. Padlan also found that in many antibodies, one or two CDRs have no amino acids in contact with the antigen (see also Vajdos et al. 2002 J Mol Biol 320: 415-428). CDR residues not in contact with the antigen can be identified by molecular modeling and / or empirically, from regions of Kabat CDRs outside of Chothia CDRs, based on past studies (for example, residues H60-H65 of CDRH2 are often not required). When a CDR or its residue is omitted, it is usually replaced with the amino acid or consensus of such a sequence at the corresponding position in another human antibody sequence. The position of substitution within the CDR, and the amino acid for substitution, can also be selected empirically.

[0115] In certain embodiments, substitutions, insertions or deletions may be present within 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 its antigen. For example, conservative changes that do not substantially reduce binding affinity (e.g., conservative substitutions provided herein) may be made within the CDRs. Such changes may be, for example, outside of antigen contact residues within the CDRs. In certain embodiments of the variant VH and VL sequences provided above, each CDR is either not changed or contains one, two, or three or fewer amino acid substitutions.

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

[0117] Amino acid sequence insertions include amino-terminal fusions and / or carboxyl-terminal fusions ranging from 1 residue in length to polypeptides containing hundreds or more residues, as well as insertions of sequences of one or more amino acid residues between. Examples of terminal insertions include antibodies having an N-terminal methionyl residue. Other insertion variants of the antibody molecule include fusions of the antibody N-terminus or antibody C-terminus to an enzyme (e.g., an enzyme for ADEPT) or a polypeptide that extends 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 development potential and are thus relatively developable.

[0119] The term "developable" refers to the extent to which one or more polypeptides among a plurality of polypeptides possess desirable properties such as, for example, desirable expression, solubility, viscosity, aggregation, chemical stability and / or physical stability, desirable shelf life, melting point, pharmacokinetic profile, circulation half-life, and clearance characteristics in mammalian cells. Such properties may be treated independently as markers, as combinations of subsets of such markers, or comprehensively, with respect to the likelihood that the one or more polypeptides can be successfully developed as therapeutic agent 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, relatively long shelf life, relatively high melting point, relatively long circulation half-life, relatively long clearance time, etc. Polypeptides having 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 circulation half-life, relatively short clearance time, etc.

[0120] For example, methods and assays that can be employed to confirm the extent to which polypeptides such as anti-CD3 antibodies and / or antigen-binding fragments thereof described herein possess desirable developability characteristics are available in the art, such as 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 measurement, whole cell binding, tissue microarray methodology, BVP ELISA assay, AC-SINS assay (Liu et al; MAbs, Vol. 6, 483-492 (2014); differential scanning calorimetry, etc. (see, for example, He et al., J. Pharm. Sci., Vol. 100(4), pp. 1330-1340 (2011); Wagner et al., Pharm. Develop. & Technol (submitted 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., 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., 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 potential are many Heavy Detected as such by interaction with a specific reagent (PSR) and thus called a "multi- Heavy specificity" polypeptide. Such multi- Heavy specificity antibodies may also be referred to as being relatively "undevelopable" or relatively "non-developable".

[0122] The "developability profile" refers to an index that can be assigned to an antibody when evaluating its developability. The developability profile is a scale or criterion by which the developability of an anti-CD3 antibody 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 containing the same. The degree of interaction may be evaluated by any number of means available in the art that provide an output value correlated with the strength or affinity of the polypeptide for the portion to which it binds. Exemplary means include, for example, flow cytometry such as FACS, ELISA, quantitative immunoaffinity assay, or immunoprecipitation assay, mammalian two-hybrid assay or yeast two-hybrid assay, and the like. In the case of FACS, as shown in the examples, the degree of interaction between a polypeptide and PSR among a plurality of polypeptides generates an average fluorescence intensity (MFI) for each detected polypeptide-PSR interaction, and then the MFIs are ordered either in ascending or descending order, thereby ranking the polypeptides among the plurality of polypeptides according to the relative degree of interaction between each detected polypeptide and PSR. By performing such ranking on the ranking of a plurality of polypeptides, polypeptides having high developability can be easily identified, and polypeptides having 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 the anti-CD3 antibody described herein against the developability of a standard (or control) antibody such as an anti-HEL antibody.

[0124] In certain embodiments, the engineered pH-dependent CD3 binding domain and antibodies containing the same of the invention may be further modified to contain additional non-proteinaceous moieties that are known in the art and readily available. Moieties suitable for derivatizing antibodies include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include polyethylene glycol (PEG), ethylene glycol / prolylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, 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-vinyl pyrrolidone) polyethylene glycol, polypropylene glycol homopolymers, polypropylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and combinations thereof. Polyethylene glycol propionaldehyde may be advantageous in manufacture, due to its stability in water. The polymer may be a polymer of any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody may vary and if multiple polymers are attached, they may be the same or different molecules. Generally, the number and / or type of polymers used for derivatization can be determined without limitation, but in accordance with considerations including the particular properties or functions of the antibody to be improved, whether the antibody derivative is to be used therapeutically under defined conditions, and the like.

[0125] In certain embodiments, the engineered pH-dependent CD3 binding domain and antibodies containing the same exhibit an enhanced developability profile. The developability profile of an anti-CD3 antibody is obtained by performing one or more of a PSR assay, SCP assay, AC-SINS, ELISA, DSF assay, Tm assay, HIC assay, CIC assay, or combinations 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 from about 0.0 to about 0.45. In some embodiments, the PSR is from about 0.0 to about 0.4. In some embodiments, the PSR is from about 0.0 to about 0.35. In some embodiments, the PSR is from about 0.0 to about 0.3. In some embodiments, the PSR is from about 0.0 to about 0.25. In some embodiments, the PSR is from about 0.0 to about 0.2. In some embodiments, the PSR is from about 0.0 to about 0.15. In some embodiments, the PSR is from 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 a 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 from about 10.5 minutes to 11.5 minutes (medium HIC score). In some embodiments, the HIC score is higher than about 11.5 minutes (high HIC score). Generally, the lower the HIC score, the more favorable the developability of the antibody.

[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%. This indicates 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, such as, for example, silent Fc.

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

[0132] The "Fc region" is the C-terminal region of an immunoglobulin heavy chain and contains at least a portion of the constant region, including the native sequence Fc region and variant Fc regions. The Fc region of a human IgG heavy chain can range from Cys226 or Pro230 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region follows the EU numbering system, also called 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 the anti-CD3 antibodies of the present disclosure to thereby create Fc region variants (see, for example, US2012 / 0251531). Fc region variants may include a human Fc region sequence (e.g., the Fc region of human IgG1, IgG2, IgG3, or IgG4) that contains an amino acid modification (e.g., substitution) at one or more amino acid positions.

[0134] In certain embodiments, the present disclosure anticipates anti-CD3 antibody variants that possess some but not all effector functions, such that the antibody half-life in vivo is important, but which are desirable candidates for applications where certain effector functions (e.g., complement and ADCC) are unnecessary or harmful. In vitro and / or in vivo cytotoxicity assays can be performed to confirm a decrease / loss of CDC activity and / or ADCC activity. For example, an Fc receptor (FcR) binding assay can be performed to confirm that the antibody lacks FcγR binding (and thus is likely to lack ADCC activity), but retains FcRn binding ability. The major cells that mediate ADCC (e.g., NK cells) express only FcγIII, while monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991) at page 464, Table 3. Non-limiting examples of in vitro assays for evaluating the ADCC activity of a target molecule are described in U.S. Patent 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); see also U.S. Patent No. 5,821,337 (Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, a non-radioactive assay method 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)). Effector cells useful in such assays include peripheral blood mononuclear cells (PBMC) and natural killer cells (NK).Alternatively, or additionally, the ADCC activity of the target molecule may be evaluated in vivo in an animal model, such as the animal models 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 cannot bind to C1q and thus lacks CDC activity. See, for example, the binding ELISAs for C1q and C3c in WO2006 / 029879 and WO2005 / 100402. A CDC assay may be performed to evaluate complement activation (see, for example, Gazzano-Santoro et al. J. Immunol Methods 202:163 (1996); Cragg, M.S. et al. Blood. 101:1045-1052 (2003); and Cragg, M.S. and M.J. Glennie Blood. 103:2738-2743 (2004)). FcRn binding and determination of in vivo clearance / half-life can also be performed using methods known in the art (see, for example, Petkova, S.B. et al. Int’l. Immunol 18(12):1759-1769 (2006)).

[0135] In some embodiments, antibodies with reduced effector function include antibodies having one or more substitutions among residues 238, 265, 269, 270, 297, 327, and 329 of the Fc region (U.S. Pat. Nos. 6,737,056 and 8,219,149). In some embodiments, Fc variants include Fc variants having substitutions at two or more of positions 265, 269, 270, 297, and 327 of the amino acid, including the so-called "DANA" Fc variant in which residues 265 and 297 are substituted with alanine (U.S. Pat. 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, coupled via, for example, 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 such that the degree to which the antibody is glycosylated is increased or decreased. Adding or removing glycosylation sites in the anti-CD3 antibodies of the present disclosure can be readily achieved by altering the amino acid sequence such that one or more glycosylation sites are created or removed.

[0139] Production of Anti-CD3 Antibodies and Antigen-Binding Fragments Thereof An anti-CD3 antibody and / or its antigen-binding fragment may be produced using recombinant methods. For example, an isolated nucleic acid encoding the anti-CD3 antibody described herein is provided. The nucleic acid may encode an amino acid sequence containing the VL of the antibody and / or an amino acid sequence containing the VH of the antibody (e.g., the light chain and / or heavy chain of the antibody). In a further embodiment, one or more vectors (e.g., expression vectors) containing such nucleic acids are provided. In a further embodiment, a host cell containing such nucleic acids is provided. In one such embodiment, the host cell contains (1) a vector containing a nucleic acid encoding an amino acid sequence containing the VL of the antibody and an amino acid sequence containing the VH of the antibody, or (2) a first vector containing a nucleic acid encoding an amino acid sequence containing the VL of the antibody and a second vector containing a nucleic acid encoding an amino acid sequence containing the VH of the antibody (e.g., transformed therewith). In one embodiment, the host cell is a eukaryotic cell such as, for example, Chinese hamster ovary (CHO) cells or lymphoid cells (e.g., Y0, NS0, Sp20 cells). In one embodiment, a method for producing an anti-CD3 antibody is provided, the method comprising culturing a host cell containing a nucleic acid encoding the antibody under conditions suitable for expression of the antibody as described above, 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 an exogenous nucleic acid has been introduced and includes the progeny cells of such cell. Host cells include transformants and transformed cells, and these cells include, regardless of the number of passages, primary transformed cells and their progeny cells derived therefrom.

[0141] Regarding the recombinant production of anti-CD3 antibodies, for example, a nucleic acid encoding the antibody is isolated as described above and inserted into one or more vectors, further cloning is performed, and / or it is expressed in a host cell. The nucleic acid can be easily isolated and sequence-analyzed by conventional methods (e.g., by using oligonucleotide probes that can specifically bind to the genes encoding the heavy and light chains of the antibody).

[0142] Suitable host cells for cloning or expressing vectors encoding antibodies include prokaryotic cells or eukaryotic cells. For example, antibodies can be produced in bacteria, particularly when glycosylation and Fc effector functions are not required. For the expression of antibody fragments and polypeptides in bacteria, see, for example, U.S. Pat. Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods in Molecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa, N.J., 2003), pp. 245-254, which describes the expression of antibody fragments in E. coli.) After expression, the antibody may be isolated from the bacterial cell paste in the soluble fraction and further purified. In addition to prokaryotes, eukaryotes such as filamentous fungi or yeasts, including fungal and yeast strains whose glycosylation pathways have been "humanized" to produce antibodies with partial or complete human glycosylation patterns, are also suitable hosts for cloning or expressing vectors encoding antibodies. See, for example, 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. Pat. 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 torso cell lines adapted to grow in suspension may be useful. Other examples of useful mammalian host cell lines include monkey kidney CV1 cell line transformed by SV40 (COS-7), 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), dog kidney cells (MDCK; buffalo rat liver cells (BRL 3A), human lung cells (W138), human liver cells (Hep G2), mouse mammary tumor (MMT 060562), TRI cells described, for example, in Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982), MRC 5 cells, and FS4 cells. 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)), as well as myeloma cell lines such as, for example, 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 (B.K.C. Lo, ed., Humana Press, Totowa, N.J.), 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 activities by 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 a hybridoma supernatant. As a control, immobilized CD3 is incubated in a solution containing the first labeled antibody but no second unlabeled antibody. After incubation under conditions where the first antibody can bind to CD3, excess unbound antibody is removed and the amount of label bound to the immobilized CD3 is measured. If the amount of label bound to the immobilized CD3 is significantly decreased in the test sample compared to the control sample, this indicates that the first and second antibodies are competing for binding to CD3. See, for example, Harlow and Lane (1988) Antibodies: A Laboratory Manual. Ch. 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.).

[0145] Anti-CD3 antibodies and / or antigen-binding fragments thereof having biological activity 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 (ex vivo). Many Heavy Specific anti-CD3 antibodies (e.g., having one arm that binds to CD3 and another arm that binds to a different target, such as a tumor antigen, cell surface antigen, etc.) HeavyIn the case of specific antibodies, etc., biological activity may include activation of effector cells (e.g., activation of CD8+ T and / or CD4+ T cells), expansion of the effector cell population (i.e., increase in the number of T cells), decrease in the target cell population (i.e., decrease in the cell population expressing the second biomolecule on the cell surface), and / or killing of target cells.

[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" encompasses quantitative or qualitative detection.

[0147] In certain embodiments, labeled anti-CD3 antibodies are provided. The anti-CD3 antibodies and / or antigen-binding fragments thereof described herein may include labels or moieties that are detected directly (e.g., fluorescent labels, dye labels, labels with high electron density, chemiluminescent labels, and radioactive labels), or indirectly (e.g., enzymes or ligands). Non-limiting examples of labels include, for example, 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. Patent No. 4,737,456), luciferin, 2,3-dihydrophthalazinedione, horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, monosaccharide oxidases such as glucose oxidase, galactose oxidase, and enzymes conjugated with hydrogen peroxide for oxidizing dye precursors such as HRP, lactoperoxidase, or microperoxidase, heterocyclic oxidases such as uricase and xanthine oxidase, biotin / avidin, spin labels, bacteriophage labels, stable free radicals, and the like.

[0148] The CD3 antibodies and / or antigen-binding fragments thereof described herein, and pharmaceutical compositions of such antibodies, may be used in methods of treatment. In one embodiment, the anti-CD3 antibodies and / or antigen-binding fragments thereof described herein, or pharmaceutical compositions comprising 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 in the treatment of 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, for example, about pH 6. Accordingly, binding and activity in and around the tumor microenvironment are promoted. In some embodiments, the use of the anti-CD 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 render a mammal susceptible 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 cancer, such as tumors.

[0151] As used herein, the term "tumor" refers to the growth and proliferation of all neoplastic cells, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.

[0152] "Cancer" refers to a physiological state in mammals characterized by uncontrolled cell growth. Examples of cancer include, but are not limited to, carcinomas, lymphomas, granulomas, sarcomas, and leukemias or lymphoid malignancies, and more specific examples include lung cancers including squamous cell carcinoma (e.g., squamous cell carcinoma), small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma, peritoneal cancer, hepatocellular carcinoma, gastric cancer (gastric cancer or stomach cancer) including gastrointestinal cancer and gastrointestinal stromal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, urinary tract cancer, 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), prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, melanoma, superficial spreading melanoma, lentigo maligna melanoma, acral lentiginous melanoma, nodular melanoma, multiple myeloma, and B cell lymphoma (including low grade / follicular non-Hodgkin lymphoma (NHL), small lymphocytic (SL) NHL, intermediate / follicular NHL, intermediate diffuse NHL, high grade immunoblastic NHL, high grade lymphoblastic NHL, high grade small non-cleaved cell NHL, large cell NHL, mantle cell lymphoma, AIDS-related lymphoma, and Waldenström macroglobulinemia), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, chronic myelogenous leukemia, and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal defective growth associated with nevus syndrome, edema (e.g., edema associated with brain tumors), Meigs syndrome, brain tumors, and head and neck cancers, and related metastases. In certain embodiments, 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 lymphoma (NHL), renal cell carcinoma, prostate cancer, liver cancer, pancreatic cancer, soft tissue sarcoma, Kaposi sarcoma, carcinoid tumor, 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. However, 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 lymphoma, but is selected from the class of mature B-cell cancers including 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), Waldenström macroglobulinemia (WM), 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, Waldenström macroglobulinemia, heavy chain disease, alpha heavy chain disease, gamma heavy chain disease, mu heavy chain disease, plasma cell myeloma, solitary plasmacytoma of bone, extramedullary plasmacytoma, extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma), nodal marginal zone lymphoma, pediatric nodal marginal zone lymphoma, pediatric follicular lymphoma, primary cutaneous follicle center cell lymphoma, T-cell / histiocyte-rich large B-cell lymphoma, primary DLBCL of the CNS, primary cutaneous DLBCL, leg 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, ALK-positive large B-cell lymphoma, plasmablastic lymphoma, large B-cell lymphoma arising in HHV8-associated multicentric Castleman disease, primary effusion lymphoma, B-cell lymphomas with intermediate characteristics between unclassifiable diffuse large B-cell lymphoma and Burkitt lymphoma, and B-cell lymphomas with intermediate characteristics between unclassifiable diffuse large B-cell lymphoma and classical Hodgkin lymphoma.

[0153] As used herein, "treatment" or "treating" or "treatment" refers to a clinical intervention in an attempt to alter the natural course of an individual being treated, and can be carried out either for prophylactic purposes or during the course of a clinical pathology. Desirable effects of treatment include, but are not limited to, prevention of the development or recurrence of a disease, 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 improvement of the prognosis.

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

[0155] As used herein, the terms "improvement" and "alleviation" refer to a decrease or reduction 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 delay the progression of a disorder or disease. As used herein, "delaying the progression" of a disorder or disease means deferring, interfering with, retarding, suppressing, stabilizing, and / or postponing the onset of the disorder or disease (e.g., a cell proliferative disorder, e.g., cancer). The delay can vary in duration depending on the disease and / or the medical history of 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 I diabetes, etc. For example, the "effective amount" of an anti-CD3 antibody disclosed herein, or a composition (e.g., a pharmaceutical composition) containing 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, such as a cell proliferative disorder like cancer, and preferably is without, or minimal with, toxic or adverse effects. The effective amount can vary depending particularly on the disease state, age, sex, and weight of the patient, as well as the ability of the antibody (or its antigen-binding fragment) to elicit a desired response in the individual, and in some instances by being co-administered with one or more additional therapeutic agents.

[0158] In some embodiments, the anti-CD3 antibody and / or its antigen-binding fragment described herein may be used to enhance immune function in an individual having a cell proliferative disorder or an autoimmune disorder. After administration, the antibody or composition can enhance immune function in an individual having a cell proliferative disorder or an autoimmune disorder by activating effector cells (e.g., T cells such as CD8+ and / or CD4+ T cells, including Tregs), expanding (increasing) the population of such effector cells, reducing the population of target cells (e.g., cells expressing a second biomolecule 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 antibody and / or its antigen-binding fragment disclosed herein can be used to treat disorders including, but not limited to, proliferative disorders, neoplastic disorders, immuno-oncological disorders, neurological disorders, cognitive disorders, neurodegenerative disorders, autoimmune disorders. In one embodiment, an effective amount of the anti-CD3 antibody can be administered to an individual having such a disorder, alone or in combination with at least one additional agent. Such "individuals" can be mammals, particularly humans.

[0160] Examples of non-limiting additional therapeutic agents include chemotherapeutic agents, antibody-drug conjugates (ADCs), and / or biological modifiers. The chemotherapeutic agent can be selected from cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP). The ADC can 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 US2014 / 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 can be selected from BCL-2 inhibitors (such as GDC-0199 / ABT-199), lenalidomide (Revlimid®), PI3K-delta inhibitors (such as idelalisib (Zydelig®)), PD-1 axis-binding antagonists, agonists such as agonistic antibodies directed to activating co-stimulatory molecules such as CD40, CD226, CD28, OX40 (such as AgonOX), GITR, CD137 (also known as TNFRSF9, 4-1BB or ILA), CD27 (such as CDX-1127), HVEM or CD127, antagonists such as antagonistic antibodies directed to inhibitory co-stimulatory molecules such as CTLA-4 (also known as CD152), PD-1, TIM-3, BTLA, VISTA, LAG-3, B7-H3, B7-H4, IDO (such as 1-methyl-D-tryptophan (also known as 1-D-MT)), TIGIT, MICA / B, GITR (such as 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 to TGF-beta such as metelimumab (also known as CAT-192), fresolimumab (also known as GC1008), LY2157299k, and adoptive immunotransfer of T cells expressing a chimeric antigen receptor (CAR) (such as cytotoxic T cells or CTLs), such as adoptive immunotransfer of T cells containing 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 an individual having such a disorder, for example, in an individual such as a human having the disorder. In one embodiment, the method of enhancing immune function activates effector cells (e.g., T cells, such as CD8+ T cells and / or CD4+ T cells), expands (increases) a population of effector cells, decreases a population of target cells, and / or administers to the individual an effective amount of an anti-CD3 antibody to kill target cells (e.g., target tumor cells).

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

[0163] In one embodiment, the pharmaceutical formulation comprises any of the anti-CD3 antibodies disclosed herein and a pharmaceutically acceptable carrier. "Pharmaceutically acceptable carrier" refers to a component in a pharmaceutical formulation other than the active ingredient that is non-toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives. In another embodiment, the 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 therapy. For example, an anti-CD3 antibody and / or its antigen-binding fragment may be co-administered with at least one additional therapeutic agent. In certain embodiments, the additional therapeutic agent is a chemotherapeutic agent, a growth inhibitor, a cytotoxic agent, an agent used in radiation therapy, an anti-angiogenic agent, an apoptosis agent, an anti-tubulin agent, or other agents such as an epidermal growth factor receptor (EGFR) antagonist (e.g., a tyrosine kinase inhibitor), a HER1 / EGFR inhibitor (e.g., erlotinib ((Tarceva (trademark)))), a platelet-derived growth factor inhibitor (e.g., Gleevec (trademark) (imatinib mesylate)), a COX-2 inhibitor (e.g., celecoxib), an interferon, a cytokine, an antibody other than the anti-CD3 antibody of the present disclosure, e.g., 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 agents.

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

[0166] The combination therapies as described above include co-administration (where two or more therapeutic agents are included in the same formulation or separate formulations), and in this case the administration of the antibodies of the present disclosure can be performed before, simultaneously with, and / or after the administration of the additional therapeutic agent. In one embodiment, the administration of the anti-CD3 antibody and the administration of the additional therapeutic agent are performed within about one month of each other, or within about one week, two weeks, or three weeks, or within about one day, two days, three days, four days, five days, or six days of each other. The anti-CD3 antibodies of the present disclosure (e.g., the bispecific anti-CD3 antibodies of the present disclosure that bind to CD3 and a second biomolecule, e.g., a cell surface antigen such as a tumor antigen, e.g., the TDB antibody of the present disclosure or a variant thereof) 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 parenterally, intraluminally, and intranasally, and, if local treatment is desired, can be administered by intralesional injection. 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 reaction in a patient than the same anti-CD3 antibody administered by intravenous injection. Depending in part on whether the administration is transient or chronic, dosing can be effected by any suitable route, such as by injection, e.g., intravenous or subcutaneous injection. A variety of dosing schedules are contemplated herein, including, but not limited to, single or multiple administrations at various times, bolus dosing, and pulse infusions.

[0168] The disclosed antibodies are formulated, dosed, and administered in a manner that is consistent with legitimate medical practice. Factors to be considered in such a situation 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 to which the agent is to be delivered, the method of administration, the dosing regimen, and other factors known to the medical practitioner. Optionally, but not necessarily, the antibody may be formulated with one or more agents currently being used to prevent or treat the disorder of interest. The effective amounts of such other agents will depend on the amount of antibody present in the formulation, the type of disorder or treatment, and other factors considered above. These are generally used at the same dosages and by the same routes of administration as those described herein, or at about 1 to 99% of the dosages described herein, or at any dosage and by any route that is empirically / clinically determined to be appropriate.

[0169] Regarding the prevention or treatment of diseases, the appropriate dosage of the disclosed antibodies (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease being treated, the type of antibody, the severity and course of the disease, whether the antibody is administered for prophylactic or therapeutic purposes, past treatments, the patient's medical history and reactivity to the antibody, as well as the discretion of the attending physician. The antibody will be appropriately administered to the patient either once or over a series of treatment courses.

[0170] As a general opinion, the therapeutically effective amount of an anti-CD3 antibody administered to a human, whether it is a single administration or multiple administrations, 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, 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 and is administered daily. In one embodiment, the 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 the first day of a 21-day cycle. The dose may be administered as a single dose, such as by infusion, or as multiple doses (e.g., 2 or 3 doses). When administered repeatedly over several days, depending on the condition, generally the treatment is continued until suppression of the desired disease symptoms appears. One exemplary dosage of the antibody is in the range of about 0.05 mg / kg to about 10 mg / kg. Thus, one or more administrations 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 dosages may be administered intermittently, for example, weekly or every 3 weeks (e.g., the patient receives about 2 to about 20 or for example about 6 administrations of the anti-CD3 antibody). A high loading dose may be administered initially, followed by one or more low loading doses. The progress of this treatment method is easily monitored by conventional techniques and assays.

[0171] In some embodiments, the disclosed method may further include additional treatment. The additional treatment may be radiotherapy, surgery, chemotherapy, gene therapy, DNA therapy, viral therapy, RNA therapy, immunotherapy, bone marrow transplantation, nanotherapy, monoclonal antibody therapy, or a combination of the foregoing. The additional treatment may be in the form of adjuvant therapy or neoadjuvant therapy. In some embodiments, the additional treatment is the administration of a small molecule enzyme inhibitor or an anti-metastatic agent. In some embodiments, the additional treatment is the administration of an agent that limits side effects (e.g., an anti-emetic agent, an agent aimed at reducing the occurrence and / or severity of side effects of treatment). In some embodiments, the additional treatment is radiotherapy. In some embodiments, the additional treatment is surgery. In some embodiments, the additional treatment is a combination of radiotherapy and surgery. In some embodiments, the additional treatment is gamma irradiation. In some embodiments, the additional treatment may be the individual administration of one or more of the aforementioned therapeutic agents.

[0172] In another aspect of the disclosure, a product is provided that contains materials useful for treating, preventing, and / or diagnosing the above-described disorders. The product 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, and the like. The container may be formed from a variety of materials such as, for example, glass or plastic. The container may hold the composition alone or in combination with another composition effective for treating, preventing, and / or diagnosing a condition and may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial having a stopper penetrable by a hypodermic needle). At least one active agent in the composition is an antibody of the disclosure. The label or package insert indicates that the composition is to be used for treating a selected condition. Further, the product may include (a) a first container containing the composition therein, the composition comprising an antibody of the disclosure, and (b) a second container containing the composition therein, the composition comprising a further cytotoxin or another therapeutic agent. The product in this embodiment of the disclosure may further include a package insert indicating that the composition can be used for treating a particular condition. Alternatively, or in addition, the product may further include a second (or third) container containing a pharmaceutically acceptable buffer such as, for example, bacteriostatic water for injection (BWFI), phosphate buffered saline, Ringer's solution, and dextrose solution. Further, other buffers, diluents, filters, needles, syringes, and other articles desirable from a commercial and user perspective may be included.

[0173] Accordingly, the manufacture and / or preparation of pharmaceutical compositions comprising anti-CD3 antibodies and / or antigen-binding fragments disclosed herein is 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 I diabetes, etc.).

[0174] In some embodiments, a pharmaceutical composition comprising an anti-CD3 antibody and / or an antigen-binding fragment thereof described herein is prepared, for example, by mixing the antibody having a desired purity with one or more optional pharmaceutically acceptable carriers (Remington’s Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)) in the form of a lyophilized formulation or an aqueous solution, and optionally prepared for modified (e.g., sustained) release. Exemplary lyophilized antibody formulations are described in U.S. Patent No. 6,267,958. Aqueous antibody formulations include those described in U.S. Patent No. 6,171,586 and WO2006 / 044908, the latter of which includes a histidine-acetic acid buffer.

[0175] Pharmaceutically acceptable carriers are generally non-toxic to the recipient at the dosages and concentrations employed, and include, but are not limited to, buffers such as phosphates, citrates, and other organic acids, antioxidants such as ascorbic acid and methionine, preservatives (e.g., octadecyl dimethyl benzyl 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, proteins such as serum albumin, gelatin, or immunoglobulins, hydrophilic polymers such as polyvinyl pyrrolidone, amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine, monosaccharides, disaccharides, and other carbohydrates such as 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 nonionic surfactants such as polyethylene glycol (PEG). In this specification, examples of pharmaceutically acceptable carriers further include, for example, soluble neutral-active hyaluronidase glycoproteins (sHASEGP), interstitial drug dispersants such as human soluble PH-20 hyaluronidase glycoprotein, such as rHuPH20 (HYLENEX®, Baxter International, Inc.). Specific exemplary sHASEGP, including rHuPH20, and methods of using the same are described in U.S. Patent Application Publication Nos. 2005 / 0260186 and 2006 / 0104968.

[0176] Such formulations may contain a plurality of active ingredients, if necessary for the particular indication to be treated, having complementary activities that do not have a detrimental effect on each other, and being present in an amount effective for the intended purpose. For example, it may be desirable to further provide additional therapeutic agents (such as chemotherapeutic agents, cytotoxic agents, growth inhibitors, and / or antihormonal agents, etc.).

[0177] The active ingredients may each be encapsulated in a colloidal drug delivery system (such as liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules, etc.), or in the state of a macroemulsion, by droplet formation technology, or by interfacial polymerization, in microcapsules prepared thereby, such as microcapsules of hydroxymethylcellulose or gelatin, and poly-(methyl methacrylate) microcapsules. 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 parental anti-CD3 antibody clone ADI-26906 (antibody number 1 in Table 1). ADI-26906 was first disclosed in PCT / US2018 / 031705 (ADI-26906 was not pH-engineered). The entire disclosure of that document is incorporated herein by reference. The following three library designs were utilized to incorporate histidine: 1) adding substitutions of single or double histidine (His) to the H3+L3 jumping double, with or without the NNK degenerate codons adjacent to His, thereby generating a theoretical diversity of 3.4×10 5 ; 2) adding an H3 jumping doublet to a pre-made H1 / H2 diversity library to generate a diversity of 6.8×10 8results in a theoretical diversity of, and 3) perform a walking singlet of H3+L3 NNK / His or His / NNK, thereby 1.2×10 5 results in a theoretical diversity of. The libraries were generated and expanded as described above (see, for example, WO2009036379; WO2010105256; WO2012009568; Xu et al., Protein Eng Des Sel. 2013 Oct;26(10):663-70). The L1 design was synthesized as a full VK using SGI BioXp (SGI-DNA, La Jolla, CA). Substitutions were limited to the CDRs. However, it is also expected that substitutions can be designed within the FRs. Sequence analysis of variants from each library showed a total of 0 to 6 His substitutions per VH or VK of the variants.

[0179] Five rounds of selection were performed using three libraries against biotinylated CD3 antigen. For the first round of selection of the H1 / H2+H3 library, magnetic bead sorting technology utilizing the Miltenyi MACS system was performed in principle as described (Siegel et al., J Immunol Methods. 2004 Mar;286(1-2):141-53). Briefly, approximately 10 9Individual yeast cells were incubated at pH 6.0 with 1 mL of 100 nM biotinylated CD3 antigen for 15 minutes at room temperature in pH 6.0 FACS wash buffer PBS containing 0.1% BSA. 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 for 15 minutes at 4°C. The yeast was 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 was eluted with 5 mL of growth medium and then grown overnight. For two libraries with low diversity, the first round of selection was performed using flow cytometry (FACS). Briefly, yeast cells (about 10 9 yeast cells / library) were incubated at pH 6.0 with 0.25 mL of 100 nM biotinylated CD3 antigen for 15 minutes at room temperature in pH 6.0 FACS wash buffer PBS containing 0.1% BSA. The yeast was washed with FACS buffer, labeled, and sorted.

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

[0181] The 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, buffer-exchanged into PBS, and then a biotinylation reagent (EZ-Link Sulfo-NHS-Biotinylation Kit, Thermo Scientific, catalog number 21425) at a molar ratio of 1:7.5 was added. The mixture was kept at 4 °C overnight and further buffer-exchanged to remove free biotin in the solution. Biotinylation was confirmed via streptavidin sensor binding of the labeled protein on ForteBio. The success of the biotinylation of the CD3 protein antigen was confirmed via detectable binding to a streptavidin-binding biosensor installed on a ForteBio Octet (trademark) Red384 Interferometer (Pall ForteBio, Menlo Park, California) according to the manufacturer's guidelines (data not shown). In CD3 pre-saturation method #1 (shown in Figure 1A), yeast cells were pre-saturated 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, the yeast cells were incubated with biotinylated CD3 antigen (shown as the starred green circles in Figure 1A) at pH 6 for 10 minutes. Control cells were incubated with biotinylated CD3 antigen at pH 7.4. Then, the binders labeled at pH 6 were sorted and characterized. In CD3 pre-saturation method #2 (shown in Figure 1B), yeast cells were pre-saturated with native CD3 antigen at pH 6.0 for 10 minutes. Next, the yeast cells were washed at pH 6.0 and incubated for 10 minutes at either pH 7.4 or pH 6.0. Finally, the yeast cells were incubated with biotinylated CD3 antigen for 10 minutes at the opposite pH (if the cells were 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).Next, binders labeled with biotinylated CD3 antigen were sorted and characterized.

[0182] Four rounds of FACS selection were performed on three libraries from MACS / FACS selection. Approximately 1 × 10 8 cells of yeast per library were pelleted, washed three times with wash buffer, incubated separately with 100 nM biotinylated CD3 antigen for at least 10 minutes at room temperature at pH 6.0 and pH 7.4, or processed through pre-saturation and pH toggling of Method #2 above. The yeast were then washed twice and stained for 15 minutes at 4°C with either a secondary reagent of goat anti-human F(ab’)2 kappa-FITC (Southern Biotech, Birmingham, Alabama, Catalog No. 2062-02) diluted 1:100, streptavidin-Alexa Fluor 633 (Life Technologies, Grand Island, New York, Catalog No. S21375) diluted 1:500, or extra avidin-phycoerythrin (Sigma-Aldrich, St. Louis, Catalog No. E4011) diluted 1:50. 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 group selected from the first round of FACS was advanced to the next round.

[0183] For the second, third, and fourth FACS rounds against the group selected above, positive sorting for CD3 binders at pH 6.0 and negative sorting to reduce binders at pH 7.4 and multispecific reagent binders were included (Xu et al., Protein Eng Des Sel. 2013 Oct;26(10):663 - 70). In the second round of FACS (R3), cells were processed through pre - saturation and pH toggling of method #2 (described above), or negatively sorted against non - binders selected at pH 7.4. In the third round of FACS (R4), the output from R3 was pooled with the output from the CD3 pre - saturation method #2 of R2, and CD3 pre - saturation method #1 was performed. In the final round of FACS (R5), the output from R4 was confirmed for PSR reactivity and CD3 binding in humans and cynomolgus (Cyno) at pH 6 and 7.4. The output of each round was seeded, and isolates were selected for sequence analysis and characterization.

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

[0185] Figure 3 shows an exemplary FACS plot for Round 3, comparing the inputs of the positive sort at pH 6.0 and the negative sort at pH 7.4 from Round 2. Briefly, the sorts from Round 2 were incubated at pH 6.0 and pH 7.4 with 100 nM HuCD3-hd. The overlay columns show that the input cell population (from the Round 2 sort) exhibits higher binding at pH 6.0 compared to pH 7.4. Using the pre-saturation / toggle method #2, the cells were carried over to the next round of selection.

[0186] Figure 4 shows exemplary FACS plots from Round 4 and Round 5. Round 4 compared cells incubated with 100 nM HuCD3-hd at pH 6 and pH 7.4. Round 4 also compared cells on which the pre-saturation / toggle method was performed 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 for CD3 The affinities of anti-CD3 antibodies for CD3 at pH 6.0 and pH 7.4 were determined on the ForteBio Octet as the rate constants (k a k d K D) was determined by measuring. ForteBio affinity measurements were generally performed 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 AHC sensors. The sensors were equilibrated offline for 30 minutes in assay buffer and then monitored online for 60 seconds for baseline establishment. For avidity binding measurements, sensors loaded with IgG were exposed to 100 nM antigen (human or cynomolgus CD3) for 3 minutes, then transferred to assay buffer for 3 minutes for dissociation rate measurement. Rate data were fit using a 1:1 binding model in data analysis software provided by ForteBio. Table 2 presents the rate constants for the selected clones. Table 3 presents the equilibrium dissociation constants (K D ) for the selected clones against human CD3 at pH 6.0 and 7.4.

[0188] The specificity of anti-CD3 antibody against 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 chilled PBSF buffer, pH 7.4 (PBS + 0.1% BSA, pH 7.4). Approximately 200,000 cells per well of a 96-well plate were dispensed and pelleted by centrifugation (500 x g for 5 minutes). The 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. 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. The 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. Then incubated in the dark on ice for 20 minutes, after which the cells were washed twice with either PBSF pH 7.4 or PBSF pH 6.0. Binding was analyzed on a FACS Canto II. The mean fluorescence intensity (MFI) at pH 6.0 and 7.4 of the selected clones 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 sort. 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 sort. The blue circles represent round 2 / 3 clones obtained via pre-saturation / toggle sort at pH 6.0, the yellow circles represent round 2 / 3 clones obtained via negative sort at pH 7.4, and the red circles represent the parental clone ADI-26906. The results indicate 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 is weak and was designated as a two-group binder. Positive selection at pH 6.0, and pre-saturation / toggle sort yielded clones with mixed selectivity but high response / affinity, which was designated as a one-group binder.

[0190] The 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). The group 2 binders can include, for example, 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), 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 ForteBio experiments for four clones compared to the parental clone ADI-26906. The K D for each clone was calculated at pH 7.4 and pH 6.0. The ratio of K D was obtained by dividing the K D at pH 7.4 by the K D 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), are stronger (lower K DIt shows that they are combined at pH 6.0. For example, some clones designated as two-group 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 region, CDRL1 region, and CDRL3 region that could cause differences in binding are highlighted in the sequence columns of Figure 6 (SEQ ID NOs: 576 to 590, in the order of appearance). Table 2 shows additional kinetic and PSR data for the 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), have low PSR scores, and provide broad affinity for CD3.

[0192] Analysis of 258 unique clones identified using the methods of the present disclosure revealed consensus motifs within the CDR regions. In some embodiments, the present disclosure provides an antibody 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 comprise 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; SAD10319_P01_H02; SAD10319_P01_B03; SAD10319_P01_C03; SAD10319_P01_D03; SAD10319_P01_F03; SAD10319_P02_A04; SAD10319_P02_C04; SAD10319_P02_E04; SAD10319_P02_F04; SAD10319_P02_A05; SAD10319_P02_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; 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; and SAD10319_P06_H10.;

[0193] In some embodiments, the present disclosure provides an antibody comprising a CDRH3 binding domain comprising a consensus motif, the consensus motif comprising the ARDX1YGX2X3X4YDX5 sequence, 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_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; and LAD9966_P01_A08.

[0194] In some embodiments, the present disclosure provides an antibody comprising a CDRH3 binding domain comprising a consensus motif, the consensus motif comprising the ARDAHX1X2YX3X4DX5 sequence, 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 comprise 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 present disclosure provides an antibody comprising a CDRH3 binding domain comprising a consensus motif, the consensus motif comprising the ARDAX1HRX2FYDV sequence, 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 comprise 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 present disclosure provides an antibody comprising a CDRH3 binding domain comprising a consensus motif, the consensus motif comprising the ARDX1YHRYFYDX2 sequence, wherein 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 present disclosure provides an antibody comprising a CDRH3 binding domain comprising a consensus motif, the consensus motif comprising the AX1DAYX2X3X4HX5DV sequence, wherein X1 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 X1, X2, X3, X4, and X5 is H. The following 14 clones comprise the following 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 present disclosure provides an antibody comprising a CDRH3 binding domain comprising a consensus motif, the consensus motif comprising the ARDX1X2GRYFYDV sequence, 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 motifs: 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 present disclosure provides an antibody comprising a CDRH3 binding domain that includes a consensus motif, the consensus motif including the ARDX1X2X3RYFYDX4 sequence, 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 include 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-48589; ADI-48652; ADI-48654; ADI-48592; ADI-57401; ADI-57406; ADI-57274; ADI-57413; ADI-57414; ADI-57415; 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; 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 present disclosure provides an antibody comprising a CDRH3 binding domain that includes a consensus motif, the consensus motif comprising the ARDX1X2X3X4YFYDX5 sequence, 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 include the following consensus motifs: 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 present disclosure provides an antibody comprising a CDRH3 that is a CD3-binding domain, the CDRH3-binding domain comprising a consensus motif, the consensus motif comprising the sequence AX1DX2X3X4X5X6X7X8DX9, where X1 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 X1, 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_P04_D11; SAD10319_P01_A01; SAD10319_P01_C01; SAD10319_P01_E01; SAD10319_P01_A02; SAD10319_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_P02_E04; SAD10319_P02_F04; SAD10319_P02_A05; SAD10319_P02_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; 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;ADI-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 present disclosure provides an antibody comprising a CDRH3 binding domain that includes a consensus motif, the consensus motif including the ARDAX1X2X3X4FYDX5 sequence, where X1 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 X1, X2, X3, and X5 is H. At least the following six clones include this consensus motif and are designated as a group 1 binder: ADI-48592, ADI-48595, ADI-48650, ADI-48652, ADI-48662, and ADI-48666.

[0203] In some embodiments, the present disclosure provides an antibody comprising a CDRH3 binding domain that includes a consensus motif, the consensus motif including 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 (SEQ ID NO: 596). In some embodiments, at least one of X1, X2, X3, X4, X5, X7, and X8 is H. At least the following 21 clones include 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 an antibody comprising a CDRH2 binding domain that includes a consensus motif, the consensus motif including the sequence WIDLENANTIYDAKFQG (SEQ ID NO: 9). The following 148 clones include 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; SAD10318_P01_G03; SAD10318_P01_H03; SAD10318_P02_B04; SAD10318_P02_C04; SAD10318_P02_D04; SAD10318_P02_E04; SAD10318_P02_G04; SAD10318_P02_H04; SAD10318_P02_A05; SAD10318_P02_B05; SAD10318_P02_C05; SAD10318_P02_D05; SAD10318_P02_F05; SAD10318_P02_G05; SAD10318_P02_H05; SAD10318_P02_B06; SAD10318_P02_D06; SAD10318_P02_E06; 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_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_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. Further, 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 present disclosure provides an antibody comprising a CDRH2 binding domain that includes a consensus motif, the consensus motif including 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 include 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; SAD10319_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 present disclosure provides an antibody comprising a CDRH2 binding domain comprising a consensus motif, the 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 comprise 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 present disclosure provides an antibody comprising a CDRH2 binding domain that includes a consensus motif, the consensus motif including 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 include 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 present disclosure provides an antibody comprising a CDRH2 binding domain that includes a consensus motif, the consensus motif including 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 7 clones include 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 present disclosure provides an antibody comprising a CDRH2 binding domain that includes a consensus motif, the consensus motif including 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 include 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 present disclosure provides an antibody comprising a CDRH2 binding domain that includes a consensus motif, the consensus motif including 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 include this consensus motif: SAD10319_P01_F03; SAD10319_P02_H05; SAD10319_P02_D06; SAD10319_P03_E08; and SAD10319_P03_H08.

[0211] In some embodiments, the present disclosure provides an antibody comprising a CDRH2 binding domain that includes a consensus motif, the consensus motif including 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 include this consensus motif: SAD10319_P03_H07; SAD10319_P04_E11; SAD10319_P04_F11; SAD10319_P04_B12; and SAD10319_P04_D12.

[0212] In some embodiments, the present disclosure provides an antibody comprising a CDRH2 binding domain that includes a consensus motif, the consensus motif including 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 include this consensus motif: SAD10319_P02_E04; SAD10319_P04_C10; and SAD10319_P04_H12.

[0213] In some embodiments, the present disclosure provides an antibody comprising a CDRH2 binding domain that includes a consensus motif, the consensus motif including 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 include this consensus motif: SAD10319_P02_B05; SAD10319_P02_B06; and SAD10319_P05_D02.

[0214] In some embodiments, the present disclosure provides an antibody comprising a CDRH2 binding domain that includes a consensus motif, the consensus motif including 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 include this consensus motif: SAD10319_P05_B03; SAD10319_P05_B04; and SAD10319_P05_D04.

[0215] In some embodiments, the present disclosure provides an antibody comprising a CDRH2 binding domain that includes a consensus motif, the consensus motif including 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 include this consensus motif: SAD10319_P01_C03; SAD10319_P03_G09; and SAD10319_P06_F10.

[0216] In some embodiments, the present disclosure provides an antibody comprising a CDRH2 binding domain that includes a consensus motif, the consensus motif including 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 include this consensus motif: SAD10319_P01_A02; SAD10319_P04_C12; and SAD10319_P05_G02.

[0217] In some embodiments, the present disclosure provides an antibody comprising a CDRH2 binding domain that includes a consensus motif, the consensus motif including the SINAGDANTKYSQKFQG sequence (SEQ ID NO: 22). The following two clones include this consensus motif: SAD10319_P04_G11 and SAD10319_P06_H07.

[0218] In some embodiments, the present disclosure provides an antibody comprising a CDRH2 binding domain that includes a consensus motif, the consensus motif including the X1IDPX2TGATX3YSQKFQG sequence, 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 include this consensus motif: SAD10319_P05_G04 and SAD10319_P06_E08.

[0219] In some embodiments, the present disclosure provides an antibody comprising a CDRH2 binding domain that includes a consensus motif, the consensus motif including the WINAGDAATVYSQKFQG sequence (SEQ ID NO: 24). The following two clones include this consensus motif: SAD10319_P06_G11 and SAD10319_P06_H11.

[0220] In some embodiments, the present disclosure provides an antibody comprising a CDRH2 binding domain that includes 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 include 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; SAD10319_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; SAD10319_P06_E09; 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;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 present disclosure provides an antibody comprising a CDRH2 binding domain that includes a consensus motif, the consensus motif including the sequence WIDAGTGX1TX2YSQKFQG, where X1 is L, F, N, or A and X2 is T or K (SEQ ID NO: 595). At least the following four clones include this consensus motif and are designated as group 2 binders: ADI-48636, ADI-48638, ADI-48624, and ADI-48635.

[0222] In some embodiments, the present disclosure provides an antibody comprising a CDRH1 binding domain that includes a consensus motif, the consensus motif including 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 include 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; SAD10318_P01_G03; SAD10318_P01_H03; SAD10318_P02_B04; SAD10318_P02_C04; SAD10318_P02_D04; SAD10318_P02_E04; SAD10318_P02_G04; SAD10318_P02_H04; SAD10318_P02_A05; SAD10318_P02_B05; SAD10318_P02_C05; SAD10318_P02_D05; SAD10318_P02_F05; SAD10318_P02_G05; SAD10318_P02_H05; SAD10318_P02_B06; SAD10318_P02_D06; SAD10318_P02_E06; 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_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_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.;

[0223] In some embodiments, the present disclosure provides an antibody comprising a CDRH1 binding domain that includes a consensus motif, the consensus motif including the YTFX1X2X3X4MH sequence, where X1 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 X1, X2, X3 and X4 is H. The following 61 clones include 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; SAD10319_P02_C04; SAD10319_P02_D04; SAD10319_P02_E04; SAD10319_P02_F04; SAD10319_P02_H04; SAD10319_P02_A05; SAD10319_P02_E05; SAD10319_P02_A06; SAD10319_P02_B06; SAD10319_P02_C06; SAD10319_P02_F06; SAD10319_P02_G06; SAD10319_P02_H06; SAD10319_P03_C07; SAD10319_P03_G07; SAD10319_P03_H07; SAD10319_P03_D08; SAD10319_P03_G08; SAD10319_P03_H08; SAD10319_P03_E09; SAD10319_P03_F09; SAD10319_P03_G09; SAD10319_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_P06_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 present disclosure provides an antibody comprising a CDRH1 binding domain that includes a consensus motif, the consensus motif including 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). In some embodiments, at least one of X1, X2, X3, and X4 is H. The following 35 clones include 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; SAD10319_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; SAD10319_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 present disclosure provides an antibody comprising a CDRH1 binding domain that includes a consensus motif, the consensus motif including the YTFTSX1X2MH sequence, 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 include 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 present disclosure provides an antibody comprising a CDRH1 binding domain that includes a consensus motif, the consensus motif including the YTFX1X2YX3MH sequence, 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 include this consensus motif: SAD10319_P02_F05; SAD10319_P02_G05; SAD10319_P02_H05; and SAD10319_P05_G01.

[0227] In some embodiments, the present disclosure provides an antibody comprising a CDRH1 binding domain that includes a consensus motif, the consensus motif including the YTFX1X2YVMH sequence, 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 include this consensus motif: SAD10319_P06_D12 and SAD10319_P06_E08.

[0228] In some embodiments, the present disclosure provides an antibody comprising a CDRH1 binding domain that includes a consensus motif, the consensus motif including the FNIKDYYMH sequence (SEQ ID NO: 47). At least the following 6 clones include this consensus motif and are designated as Group 1 binders: ADI-48592, ADI-48595, ADI-48650, ADI-48652, ADI-48662, and ADI-48666. Further, at least the following 16 clones include 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 present disclosure provides an antibody comprising a CDRH1 binding domain that includes a consensus motif, the consensus motif including the YTFX1X2YX3MH sequence, where X1 is E, S, or T, X2 is S or D, and X3 is A or D (SEQ ID NO: 31). At least the following 5 clones include 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 present disclosure provides an antibody comprising a CDRL3 binding domain that includes a consensus motif, the consensus motif including 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 include 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; SAD10318_P01_G02; SAD10318_P01_A03; SAD10318_P01_C03; SAD10318_P01_D03; SAD10318_P01_E03; SAD10318_P01_F03; SAD10318_P02_B04; SAD10318_P02_C04; SAD10318_P02_D04; SAD10318_P02_E04; SAD10318_P02_H04; SAD10318_P02_A05; SAD10318_P02_B05; SAD10318_P02_C05; SAD10318_P02_F05; SAD10318_P02_G05; SAD10318_P02_H05; SAD10318_P02_D06; SAD10318_P02_E06; SAD10318_P02_G06; SAD10318_P03_E07; SAD10318_P03_F07; 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_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_F11; SAD10319_P04_G11; SAD10319_P04_B12; SAD10319_P04_C12; SAD10319_P04_D12; SAD10319_P04_E12; SAD10319_P04_F12; SAD10319_P04_H12; SAD10320_P01_D01; SAD10320_P01_F01; SAD10320_P01_C02; SAD10320_P01_E02; SAD10320_P01_B03; SAD10320_P01_H03; SAD10320_P02_C04; 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 present disclosure provides an antibody comprising a CDRL3 binding domain that includes a consensus motif, the consensus motif including the KQSYX1X2RT sequence, 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 include 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_G01; 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; SAD10320_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 present disclosure provides an antibody comprising a CDRL3 binding domain comprising a consensus motif, the consensus motif comprising the X1QSX2HX3RT sequence, 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 comprise 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 present disclosure provides an antibody comprising a CDRL3 binding domain comprising a consensus motif, the consensus motif comprising the KQSX1X2X3RT sequence, 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 comprise 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 present disclosure provides an antibody comprising a CDRL3 binding domain that includes a consensus motif, the consensus motif including the KQSX1X2X3RT sequence, 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 the following 6 clones include 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 present disclosure provides an antibody comprising a CDRL3 binding domain that includes a consensus motif, the consensus motif including the X1QSX2X3X4RT sequence, 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 include 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 an antibody comprising a CDRL2 binding domain that includes a consensus motif, the consensus motif including the WASTRES sequence (SEQ ID NO: 37). The following 215 clones include 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; SAD10318_P01_G03; SAD10318_P01_H03; SAD10318_P02_B04; SAD10318_P02_C04; SAD10318_P02_D04; SAD10318_P02_E04; SAD10318_P02_G04; SAD10318_P02_H04; SAD10318_P02_A05; SAD10318_P02_B05; SAD10318_P02_C05; SAD10318_P02_D05; SAD10318_P02_F05; SAD10318_P02_G05; SAD10318_P02_H05; SAD10318_P02_B06; SAD10318_P02_D06; SAD10318_P02_E06; 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_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. Further, at least the following 6 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. And 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 present disclosure provides an antibody comprising a CDRL1 binding domain that includes a consensus motif, the 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 include 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; SAD10318_P01_F02; SAD10318_P01_G02; SAD10318_P01_A03; SAD10318_P01_C03; SAD10318_P01_D03; SAD10318_P01_E03; SAD10318_P01_F03; SAD10318_P01_G03; SAD10318_P01_H03; SAD10318_P02_B04; SAD10318_P02_C04; SAD10318_P02_D04; SAD10318_P02_E04; SAD10318_P02_G04; SAD10318_P02_H04; SAD10318_P02_A05; SAD10318_P02_B05; SAD10318_P02_C05; SAD10318_P02_D05; SAD10318_P02_F05; SAD10318_P02_H05; SAD10318_P02_B06; SAD10318_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_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_H06; SAD10320_P03_B07; SAD10320_P03_E07; 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; 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; LAD9954_P01_B02; LAD9955_P01_G02; LAD9963_P01_E06; and LAD9966_P01_A08.;

[0238] In some embodiments, the present disclosure provides an antibody comprising a CDRL1 binding domain comprising a consensus motif, the 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 present disclosure provides an antibody comprising a CDRL1 binding domain comprising a consensus motif, the consensus motif comprising the sequence KSSQSLLNASTAKNYLA (SEQ ID NO: 40) or KSSQSLLNARTRTNYLA (SEQ ID NO: 41).

[0240] In some embodiments, the present disclosure provides an antibody 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 present disclosure provides an antibody comprising a CDRL1 binding domain that includes a consensus motif, the consensus motif including 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 the following six clones include 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 present disclosure provides an antibody comprising a CDRL1 binding domain that includes a consensus motif, the consensus motif including 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 twenty-one clones include 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 present disclosure provides an antibody comprising a CDRH3 binding domain comprising a consensus motif, the consensus motif comprising the AX1DX2YX3HX4FYDV sequence, 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 a consensus motif, the consensus motif comprising the WIDLENANTIYDAKFQG sequence (SEQ ID NO: 9); the WINPX1TGATX2YSQKFQG sequence, where X1 is S, D, A, N, L or Q and X2 is V, T, D, Y or K (SEQ ID NO: 45); or the X1IDAGTGATX2YSQKFQG sequence, where X1 is W, S or D and X2 is A, H, K, T or D (SEQ ID NO: 46); a CDRH1 binding domain comprising a consensus motif, the consensus motif comprising the FNIKDYYMH sequence (SEQ ID NO: 47); a CDRL3 binding domain comprising a consensus motif, the consensus motif comprising the KSQYX1X2RT sequence, where 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, the consensus motif comprising the WASTRES sequence (SEQ ID NO: 37); and a CDRL1 binding domain comprising a consensus motif, the consensus motif comprising the KSSQSLLNARTGKNYLA sequence (SEQ ID NO: 49).

[0244] In some embodiments, the present disclosure provides an antibody comprising a CDRH3 binding domain comprising a consensus motif, the consensus motif comprising the ARDX1YGX2X3X4YDX5 sequence, 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, the consensus motif comprising the WIDLENANTIYDAKFQG sequence (SEQ ID NO: 9) or the WIX1AGTGATX2YSQKGQG sequence, 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, the consensus motif comprising the FNIKDYYMH sequence (SEQ ID NO: 47) or the YTFX1X2YX3MH sequence, where 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 KQSX1SRRT sequence, where X1 is H or Y (SEQ ID NO: 52); a CDRH2 binding domain comprising a consensus motif, the consensus motif comprising the WASTRES sequence (SEQ ID NO: 37); and a CDRL1 binding domain comprising a consensus motif, the consensus motif comprising the KSSQSLLX1AX2TX3X4NX5LA sequence, 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 present disclosure provides an antibody comprising a CDRH3 binding domain comprising a consensus motif, the consensus motif comprising the ARDAHX1X2YX3X4DX5 sequence, 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 WIDLENANTIYDAKFQG sequence (SEQ ID NO: 9); a CDRH1 binding domain comprising a consensus motif, the consensus motif comprising the FNIKDYYMH sequence (SEQ ID NO: 47); a CDRL3 binding domain comprising a consensus motif, the consensus motif comprising the KQSYSRRT sequence (SEQ ID NO: 54); a CDRH2 binding domain comprising a consensus motif, the consensus motif comprising the WASTRES sequence (SEQ ID NO: 37); and a CDRL1 binding domain comprising a consensus motif, the consensus motif comprising the KSSQSLLNAX1TGX2NX3LA sequence, 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 present disclosure provides an antibody or antigen-binding fragment comprising a CDRH3 binding domain comprising a consensus motif, wherein the consensus motif comprises the ARDAX1X2X3X4FYDX5 sequence, where X1 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 X1, X2, X3, and X5 is H (SEQ ID NO: 593); a CDRH2 binding domain comprising a consensus motif, wherein the consensus motif comprises the WIDLENANTIYDAKFQG sequence (SEQ ID NO: 9); a CDRH1 binding domain comprising a consensus motif, wherein the consensus motif comprises the FNIKDYYMH sequence (SEQ ID NO: 47); a CDRL3 binding domain comprising a consensus motif, wherein the consensus motif comprises the KQSX1X2X3RT sequence, where X1 is H or Y; X2 is T, S, or Q; 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 a consensus motif, wherein the consensus motif comprises the WASTRES sequence (SEQ ID NO: 37); and / or a CDRL1 binding domain comprising a consensus motif, wherein the consensus motif comprises the KSSQSLLNX1X2TGX3NYLA sequence, 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 H (SEQ ID NO: 594). In some embodiments, the antibody or antigen-binding fragment is designated as a Group 1 binder and comprises a CD3 binding domain selected from ADI-48592, ADI-48595, ADI-48650, ADI-48652, ADI-48662, and ADI-48666.

[0247] In some embodiments, the present disclosure provides a CDRH3 binding domain comprising a consensus motif, wherein the consensus motif comprises 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, 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, wherein the consensus motif comprises 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, wherein the consensus motif comprises the sequence FNIKDYYMH (SEQ ID NO: 47) or 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); a CDRL3 binding domain comprising a consensus motif, wherein the consensus motif comprises the sequence X1QSX2X3X4RT, where X1 is K or H, X2 is Y or H, X3 is S, H, L, V or K, 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 comprising a consensus motif, wherein the consensus motif comprises the sequence WASTRES (SEQ ID NO: 37); a CDRL1 binding domain comprising a consensus motif, wherein the consensus motif comprises 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 isProvided is an antibody or antigen-binding fragment thereof that includes a moiety that is H (SEQ ID NO: 597). In some embodiments, the antibody or antigen-binding fragment is designated as a two-group binder that includes 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] Materials and Methods In addition to the above description, the following materials and methods were employed in this example.

[0249] Production of Hu and Cy CD3εδFc heterodimer antigens. Recombinant heterodimeric CD3 Fc fusion antigens were produced in HEK293 cells by co-transfecting 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. The clarified culture supernatant was purified by Ni Sepharose 6 Fast Flow (GE Healthcare Life Sciences), removing the CD3εεFc-HIS homodimer. The CD3εδFc-HIS heterodimer was separated from the CD3δδFc-HIS homodimer by Mono Q 10 / 100 GL with a Tris-buffered KCl linear gradient at pH 8.5.

[0250] Peptide. The C-terminally biotinylated CD3ε N-terminal peptide was obtained from New England Peptide. All peptides were delivered with a purity of 95% or higher. The 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 CD3εN27 peptide has the sequence H2N-QDGNEEMGSITQTPYQVSISGTTVILT[K / SCBiot(dPEG4)]-amide (SEQ ID NO: 56), and the CD3εN13 peptide has the sequence H2N-QDGNEEMGGITQT[K / SCBiot(dPEG4)]-amide (SEQ ID NO: 57).

[0251] Biotinylation of antigen. The CD3 antigen was biotinylated using the Pierce EZ-Link Sulfo-NHS-Biotinylation Kit. Goat anti-human F(ab’)2 kappa-FITC (LC-FITC), extra avidin-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] Amplification of cell lines 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. The cells were centrifuged at 4°C, 500×g for 5 minutes. They were then resuspended in 100 μl of 100 nM IgG1 and incubated at room temperature for 20 minutes. The cells were then washed three times with buffer (phosphate - buffered saline (PBS) / 0.1% bovine serum albumin (BSA)) and resuspended in secondary reagent, typically goat anti - human R - PE (Southern Biotech). The plate was analyzed on a FACSCanto (BD Biosciences) using an HTS sample injector. Flow cytometry data was analyzed against the median fluorescence intensity in the R - PE channel.

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

[0255] Yeast production and purification of antibodies. Yeast clones were grown to saturation and then induced with shaking at 30 °C for 48 hours. After induction, yeast cells were precipitated 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 performed by subcloning the antibody into a new expression vector and then performing transient transfection and expressing it in HEK293 ADI1. Monoclonal cell lines derived from HEK293 (DSMZ) were selected for non-aggregating growth, growth rate, and transfection efficiency. Briefly, an expression vector containing the target antibody was transfected by complexation with a transfection reagent, followed by exposure to HEK cells for 1 hour, after which the culture medium was diluted to a final density of 4 million cells per mL. The cells were then cultured for 7 days with replacement of the fresh feed medium every 48 hours. After 7 days, the supernatant was collected after centrifugation and purified using protein A. If necessary, CHT column purification was added to make the monomer more than 95%.

[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). The cells were washed either with 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 the 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. The 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. Then incubated in the dark on ice for 20 minutes, after which the 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 AHC sensors. The sensors were equilibrated offline for 30 minutes in assay buffer and then monitored online for 60 seconds to establish a baseline. Sensors loaded with IgG were exposed to 100 nM antigen (e.g., CD3) for 5 minutes and then transferred to assay buffer for 5 minutes for dissociation rate measurements. Reaction rates were analyzed using a 1:1 binding model.

[0259] PSR Preparation. The multispecific 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 the starting material. In 500 mL centrifuge bottles filled up to 400 mL, the cells were precipitated at 2,400 xg for 5 minutes. The cell pellet was combined and then resuspended in 25 ml of Buffer B and precipitated at 2,400 xg for 3 minutes. The buffer was decanted and the wash was repeated once more. The cell pellet was resuspended in 3 volumes of Buffer B, which is 3 times the volume of the pellet, containing 1x protease inhibitor (Roche, Complete, EDTA-free), while maintaining the cells on ice, using a Polytron homogenizer. The homogenate was then centrifuged at 2,400 x g for 5 minutes, and the supernatant was retained and precipitated once more (2,400 x g / 5 minutes) to ensure the removal of unbroken cells, cell debris, and nuclei. The resulting supernatant is the total protein preparation. The supernatant was then transferred to two Nalgene Oak Ridge 45 mL centrifuge tubes and precipitated at 40,000 x g, 4°C for 40 minutes. The supernatant containing the separated cytosolic proteins (SCP) was then transferred to a clean Oak Ridge tube and centrifuged once more at 40,000 x g. In parallel, the pellet containing the membrane fraction (EMF) was retained and centrifuged at 40,000 for 20 minutes to remove the residual supernatant. The EMF pellet was then rinsed with Buffer B. Then 8 mL of Buffer B was added to the membrane pellet to detach the pellet and transferred to a Dounce Homogenizer. After the pellet was homogenized, they were transferred to 50 mL conical tubes to obtain the final EMF preparation.

[0260] Approximately 10 6 ~10 7One billion mammalian cells (e.g., CHO, HEK293, Sf9, etc.) per mL were transferred from the tissue culture environment into 4 x 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 pooled 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 CaCl2, 5 mM KCl, 5 mM MgCl2, 10% glycerol, pH 7.2) and pelleted at 550 x g for 3 minutes. The supernatant of buffer B was decanted and the cells were resuspended in 3-fold pellet volume of buffer B with 2.5x protease inhibitor (Roche, cOmplete, EDTA-free). Thereafter, protease inhibitor was included in buffer B. The cells were homogenized 4 times with 30-second pulses (Polytron homogenizer, PT1200E) and the membrane fraction was pelleted at 40,000 x g for 1 hour at 4 °C. The pellet was rinsed with 1 mL of buffer B. The supernatant was retained and designated as s. The pellet was transferred to a Dounce homogenizer with 3 mL of buffer B and resuspended by slowly moving the pestle up and down for 30 - 35 strokes. The enriched membrane fraction (EMF) was transferred to a new collection tube and the pestle was rinsed to collect all possible proteins. The protein concentration of the purified EMF was determined using the Dc-protein assay kit (BioRad). To solubilize the EMF, it was transferred to solubilization buffer (50 mM HEPES, 0.15 M NaCl, 2 mM CaCl2, 5 mM KCl, 5 mM MgCl2, 1% n-dodecyl-β-D-maltopyranoside (DDM), 1x protease inhibitor, pH 7.2) to a final concentration of 1 mg / mL. 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 is the soluble membrane protein (SMP), was collected and the protein production was quantified as described above.

[0261] Regarding biotinylation, prepare an NHS-LC-biotin stock solution according to the manufacturer's protocol (Pierce, Thermo Fisher). Briefly, add 20 μl of biotin reagent per 1 mg of EMF sample, and incubate at 4 °C for 3 hours with gentle stirring. Adjust the volume to 25 mL using Buffer B and transfer to an Oak Ridge centrifuge tube. Precipitate biotinylated EMF (b-EMF) at 40,000 × g for 1 hour and rinse the precipitate twice without disturbing it using 3 mL of Buffer C (Buffer B minus glycerol). Remove the residual solution. As described above, resuspend the precipitate in 3 mL of Buffer C using a Dounce homogenizer. The resuspended precipitate is biotinylated EMF (b-EMF) at this point. Solubilize as described above to prepare 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 presented on yeast, 2 million IgG-presenting yeasts were transferred to a 96-well assay plate, pelleted at 3000 x g for 3 minutes, and the supernatant was removed. The pellet was resuspended in a 1:10 dilution of 50 μl 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 the pellet was resuspended in 50 μl of secondary labeling mix (extra avidin-R-PE, anti-human LC-FITC and propidium iodide). This mixture was incubated on ice for 20 minutes and then washed twice with 200 μl of ice-cold PBSF. The cells were resuspended in 100 μl of ice-cold PBSF and performed on the plate on a FACSCanto (BD Biosciences) using an HTS sample injector. Flow cytometry data were analyzed for mean fluorescence intensity in the R-PE channel and normalized against appropriate controls for assessment of non-specific binding. Numerous methods for presenting or displaying antibodies or antibody fragments on the yeast surface have been reported in the past, all of which are compatible with the 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 U S A., 106(31):12891-6(2009)).

[0263] ForteBio Dynamics. The FortBio Octet HTX instrument was used in 12-channel mode (8 sensors per channel, 96 sensors per experiment) with either AHC sensors, SA sensors, or AHQ sensors. The instrument was operated with software (versions 8.2 and 9.0) supplied by the manufacturer. The sample name and concentration were entered on the plate data page, and the protein bound to the sensor was identified in the "Info" column on the sensor data page. Kinetic experiments were collected at either a reference time of 90 seconds or 180 seconds, a binding phase of 180 seconds, and a dissociation phase of 180 seconds. All files were saved in a shared network drive using a naming convention that identifies the experiment format.

[0264] HIC. IgG1 samples were buffer-exchanged to 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). The salt gradient was established on a Dionex ProPac HIC-10 column from 1.8 M ammonium sulfate, 0.1 M sodium phosphate at pH 6.5 to the same conditions without ammonium sulfate. The gradient was run at a flow rate of 0.75 ml / min for 17 minutes. An acetonitrile wash step was added at the end of the run to remove all remaining protein, and the column was re-equilibrated with more than 7 column volumes before the next injection cycle. The peak retention time was 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 middle-down LCMS analysis was performed on a Bruker maXis4G mass spectrometer coupled with an Agilent 1100 HPLC (Agilent). A POROS R2 10μm (2.1x30mm) reversed-phase column was used to remove salts in the sample. With a high-speed LC flow of 2 mL / min, separation of the sample and salts was possible, and elution of the sample and regeneration of the column were completed within a 2.1-minute cycle. Using a T-junction, a sample flow of only 0.15 mL / min was delivered to the mass spectrometer for sample analysis. The Bruker maXis 4G mass spectrometer was operated in positive ion mode and detected in the range of 750 - 2500 m / z. The remaining source parameters were set as follows: the capillary was set at 5500 V, the nebulizer was set at 4.0 bar, the drying gas was 4.0 l / min, and the drying temperature was set at 200 °C.

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

[0267] A simplified sequence listing is presented in Table 1 below. The simplified sequence listing presents the amino acid sequences of the heavy chain variable regions (HC), with each of the heavy chain variable region CDRs underlined. The amino acid sequences of the light chain variable regions (LC) are also presented, 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. An anti-cluster of differentiation 3 (CD3) antibody or an antigen-binding antibody fragment thereof, comprising: (A) a heavy chain variable region (VH) comprising CDRH1, CDRH2 and CDRH3, and (B) a light chain variable region (VL) comprising CDRL1, CDRL2 and CDRL3, wherein (I) said CDRH1, CDRH2 and CDRH3 each comprise the amino acid sequences of heavy chain CDR1, CDR2 and CDR3 respectively encompassed by SEQ ID NO: 130, and said CDRL1, CDRL2 and CDRL3 each comprise the amino acid sequences of light chain CDR1, CDR2 and CDR3 respectively encompassed by SEQ ID NO: 131; or, (II) said CDRH1, CDRH2 and CDRH3 each comprise the amino acid sequences of heavy chain CDR1, CDR2 and CDR3 respectively encompassed by SEQ ID NO: 450, and said CDRL1, CDRL2 and CDRL3 each comprise the amino acid sequences of light chain CDR1, CDR2 and CDR3 respectively encompassed by SEQ ID NO: 451, an anti-CD3 antibody or an antigen-binding antibody fragment thereof.

2. (i) CDRH1 comprises the amino acid sequence FNIKDYYMH (SEQ ID NO: 47); (ii) CDRH2 comprises the amino acid sequence WIDLENANTIYDAKFQG (SEQ ID NO: 9); (iii) CDRH3 comprises the amino acid sequence ARDX １ YHRYFYDX ２ (SEQ ID NO: 5), where X １ is H, and X ２ is V; and (iv) CDRL2 comprises the amino acid sequence WASTRAS (SEQ ID NO: 37); and, in the case of (I), (v) CDRL1 comprises the amino acid sequence KSSQSLNX １ X ２ TGX ３ NYLA (SEQ ID NO: 594), where X １ is A, X ２ is R, and X ３ is H, and, (vi) CDRL3 contains the amino acid sequence KQSX １ X ２ X ３ RT (SEQ ID NO: 36), where X １ is H, X ２ is S, and X ３ is H; or in the case of (II), (v) CDRL1 contains the amino acid sequence KSSQSLNX １ X ２ TGX ３ NYLA (SEQ ID NO: 594), where X １ is A, X ２ is H, and X ３ is H, and (vi) CDRL3 contains the amino acid sequence X １ QSX ２ X ３ X ４ RT (SEQ ID NO: 598), where X １ is H, X ２ is Y, X ３ is S, and X ４ is H, The anti-CD3 antibody or antigen-binding antibody fragment thereof according to claim 1.

3. In the case of (I), (A) VH contains an amino acid sequence having at least 90% or at least 95% identity with SEQ ID NO: 130, and (B) VL contains an amino acid sequence having at least 90% or at least 95% identity with SEQ ID NO: 131; or, In the case of (II), (A) VH contains an amino acid sequence having at least 90% or at least 95% identity with SEQ ID NO: 450, and (B) VL contains an amino acid sequence having at least 90% or at least 95% identity with SEQ ID NO: 451, The anti-CD3 antibody or antigen-binding antibody fragment thereof according to claim 1 or 2.

4. The anti-CD3 antibody or antigen-binding antibody fragment thereof according to any one of claims 1 to 3, wherein VH and VL each contain the amino acid sequences of SEQ ID NOs: 130 and 131.

5. The anti-CD3 antibody or antigen-binding antibody fragment thereof according to any one of claims 1 to 3, wherein VH and VL each contain the amino acid sequences of SEQ ID NOs: 450 and 451.

6. The anti-CD3 antibody or antigen-binding antibody fragment thereof according to any one of claims 1 to 5, comprising scFv, Fv, Fab, Fab’, Fab’-SH, F(ab’)2, diabody, scFv2-Fc2 or scFv-IgG.

7. (A) A heavy chain comprising VH and a heavy chain constant region (CH), and (B) A light chain comprising VL and a light chain constant region (CL) The anti-CD3 antibody or antigen-binding antibody fragment thereof according to any one of claims 1 to 6.

8. The anti-CD3 antibody or antigen-binding antibody fragment thereof according to claim 7, comprising an immunoglobulin molecule containing two heavy chains and two light chains interconnected by disulfide bonds or a multimer of the immunoglobulin molecule.

9. The anti-CD3 antibody or antigen-binding antibody fragment thereof according to claim 8, comprising an IgG antibody, IgA antibody, IgD antibody, IgE antibody or IgM antibody.

10. The anti-CD3 antibody or antigen-binding antibody fragment thereof according to claim 8, comprising an IgG1 antibody, IgG2 antibody, IgG3 antibody, IgG4 antibody, IgA1 antibody or IgA2 antibody.

11. The anti-CD3 antibody or antigen-binding antibody fragment thereof according to any one of claims 1 to 10, which is contained in a chimeric antigen receptor (CAR).

12. The anti-CD3 antibody or antigen-binding antibody fragment thereof according to claim 11, wherein the CAR comprises at least one transmembrane domain, at least one intracellular domain derived from a T cell receptor, and at least one co-stimulatory domain.

13. The anti-CD3 antibody or antigen-binding antibody fragment thereof according to claim 12, wherein the at least one transmembrane domain is a CD3ζ subunit.

14. The anti-CD3 antibody or antigen-binding antibody fragment thereof according to any one of claims 1 to 13, comprising a multispecific antibody or a bispecific antibody.

15. (a) a tumor target, an immuno-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 blood disease target; (b) 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, adrenomedullin, aFGF, ALCAM, ALK, ALK - 1, ALK - 7, alpha - 1 - 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 stimulator (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, CIO, CA125, CAD - 8, calcitonin, cAMP, carcinoembryonic antigen (CEA), tumor - associated antigen, cathepsin A, cathepsin B, cathepsin C / DPP I, 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, welchii 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, digitoxin, DNAM-1, Dnase, Dpp, DPPIV / CD26, Dtk, E-cad, EDA, EDA-A1, EDA-A2, EDAR, EGF, EGFR (ErbB-1), EMA, EMMPRIN, ENA, endothelin receptor, enkephalinase, eNOS, Eot, eotaxin1, EpCAM, Ephrin B2 / EphB4, EPO, ERCC, E-selectin, ET-1, factor IIa, factor VII, factor VIIIc, factor IX, fibroblast activation protein (FAP), Fas, FcR1, 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, GFRa-1, GFR-alpha1, GFR-alpha2, GFR-alpha3, GITR, glucagon, Glut 4, glycoprotein IIb / IIIa (GP IIb / 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: Hemopoietic growth factor), Hep B gp120, heparanase, Her2, Her2 / neu (ErbB-2), Her3 (ErbB-3), Her4 (ErbB-4), herpes simplex virus (HSV) gB glycoprotein, HSV gD glycoprotein, HGFA, high molecular melanoma-associated antigen (HMW-MAA: High molecular weight melanoma-associated antigen), HIV gp120, 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, IFNγ, 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 alpha2, integrin alpha3, integrin alpha4, integrin alpha4 / beta1, integrin, alpha4 / beta7, integrin alpha5 (alphaV), integrin alpha5 / beta1, integrin alpha5 / beta3, integrin alpha6, integrin beta1, integrin beta2, 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, lung surfactant, luteinizing hormone, lymphotoxin beta receptor, Mac-1, MAdCAM, MAG, MAP2, MARC, MCAM, MCAM, MCK-2, MCP, M-CSF, MDC, Mer, metalloprotease, 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 (Muc1), MUC18, Müllerian inhibiting factor, Mug, MuSK, NAIP, NAP, NCad, N-cadherin, NCAM90, NCAM, neprilysin, neurotrophin-3, -4, or -6, neurulin, nerve growth factor (NGF), NGFR, NGF-beta, nNOS, NO, NOS, Npn, NRG-3, NT, NTN, OB, OGG1, OPG, OPN, OSM, OX40L, OX40R, p150, p95, PARPr, 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), PlGF, PLP, PP14, proinsulin, prolactin, 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, TGF-beta RI (ALK-5), TGF-beta RII, TGF-beta Rllb, TGF-beta RIII, 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 Ap-2, DR4), TNFRSFIOB (TRAIL R2 DR5, KILLER, TRICK-2A, TRICK-B), TNFRSF10C (TRAIL R3 DcR1, LIT, TRID), TNFRSF10D (TRAIL R4 DcR2, TRUDD), TNFRSF11A (RANK ODF R, TRANCE R), TNFRSFllB (OPG OCIF, TR1), TNFRSF12 (T WEAK RFN14), TNFRSF13B (TACI), TNFRSF13C (BAFF R), TNFRSF14 (HVEM ATAR, HveA, LIGHT R, TR2), TNFRSF16 (NGFR p75NTR), TNFRSF17 (BCMA), TNFRSF 18 (GITR AITR), TNFRSF19 (TROY TAJ, 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 Ap o-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 Ap o-3, LARD, TR-3, TRAMP, WSL-1), TNFSF10 (TRAIL Ap o-2 ligand, TL2), TNFSF11 (TRANCE / RANK ligand ODF, OPG ligand), TNFSF12 (TWEAK Ap o-3 ligand, DR3 ligand), TNFSF13 (APRIL TALL2), TNFSF13B (BAFF BLYS, TALL1, THANKS, TNFSF20), TNFSF14 (LIGHT HVEM ligand, LTg), TNFSF15 (TLIA / VEGI), TNFSF18 (GITR ligand AITR ligand, TL6), TNFSFIA (TNF-a Connectin, DIF, TNFSF2), TNFSF1B (TNF-b LTa, TNFSF1), TNFSF3 (LTb TNFC, p33), TNFSF4 (OX40 ligand gp34, TXGP1), TNFSF5 (CD40 ligand CD154, gp39, HIM1, IMD3, TRAP), TNFSF6 (Fas ligand Ap o-1 ligand, APT1 ligand), TNFSF7 (CD27 ligandCD70), TNFRSF8 (CD30 ligand CD153), TNFRSF9 (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 CA125, tumor-associated antigen expressing Lewis Y-related sugar, TWEAK, TXB2, Ung, uPAR, uPAR-1, urokinase, VCAM, VCAM-1, VECAD, VE-cadherin, VE-cadherin-2, VEGFR-1 (flt-1), VEGF, VEGFR, VEGFR-3 (flt-4), VEGI, VFM, viral antigen, VLA, VLA-1, VLA-4, VNR integrin, von Willebrand 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 receptor, and growth factor, an antigen selected from the group consisting of; and / or, (c) an antigen 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 gene-3), TIM-3, CD20, CD2, CD19, Her2, EGFR, EpCAM, FcγRIIIa (CD16), FcγRIIa (CD32a), FcγRIIb (CD32b), FcγRI (CD64), Toll-like receptors (TLRs), TLR4, TLR9, cytokines, IL-2, IL-5, IL-13, IL-6, IL-17, IL-12, IL-23, TNFα, TGFβ, cytokine receptors, IL-2R, chemokines, chemokine receptors, growth factors, VEGF and HGF comprising at least a second antigen-binding domain that specifically binds to The anti-CD3 antibody or antigen-binding antibody fragment thereof according to claim 14.

16. (a) scFv, scFv2-Fc2 and / or scFv-IgG; (b) IgG constant domain, and / or (c) a format of multispecificity selected from the group consisting of Fab-Fc-scFv, "plug-and-play type", 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 The anti-CD3 antibody or antigen-binding antibody fragment thereof according to claim 14 or 15, comprising

17. The anti-CD3 antibody or antigen-binding antibody fragment thereof according to any one of claims 1 to 16, which binds to CD3 or CD3-expressing cells with higher binding affinity at pH 6.0 than at pH 7.

4.

18. An isolated nucleic acid or recombinant nucleic acid encoding the anti-CD3 antibody or antigen-binding antibody fragment thereof according to any one of claims 1 to 17.

19. An expression vector containing the isolated nucleic acid or recombinant nucleic acid according to claim 18.

20. A host cell transfected, transformed or transduced with the nucleic acid according to claim 18 or the expression vector according to claim 19.

21. The host cell according to claim 20, which is a eukaryotic cell.

22. The host cell according to claim 21, which is a mammalian cell or a yeast cell.

23. The host cell according to claim 22, which is a Chinese hamster ovary (CHO) cell, a human embryonic kidney (HEK) cell line or a lymphoid cell.

24. The host cell according to claim 20, which is a prokaryotic cell.

25. The host cell according to claim 24, which is a bacterial cell.

26. A pharmaceutical composition comprising the anti-CD3 antibody or antigen-binding antibody fragment thereof according to any one of claims 1 to 17, or the host cell according to any one of claims 20 to 25, and a pharmaceutically acceptable carrier and / or excipient.

27. The pharmaceutical composition according to claim 26 for use in treating a disorder in a mammal in need of such treatment or for use in the manufacture of a medicament for treating a disorder in such a mammal, wherein the disorder is a proliferative disorder, a neoplastic disorder, an immuno-oncological disorder, a neurological disorder, a neurodegenerative disorder, or an autoimmune disorder. **Claim 28** The pharmaceutical composition according to claim 27, wherein the host cell is a T cell or an NK cell. **Claim 29** The pharmaceutical composition according to claim 27 or 28, further comprising an additional therapeutic agent or used in combination with an additional therapeutic agent. **Claim 30** The pharmaceutical composition according to any one of claims 27 to 29, wherein the mammal is a human.

Images