Engineered ph-dependent anti-CD3 antibodies and methods of production and use thereof

Abstract

Provided are engineered pH-dependent anti-CD3 binding domains, and antibodies and / or antigen binding domains comprising the same, including multispecific antibodies, having, inter alia, desirable T cell activation and (re) guided target cell killing potency and developable profiles; as well as methods of their identification, isolation and production; and methods of making and using the same.

Description

[0001] This application is a divisional application of Chinese patent application No. 202080033843.5, filed on June 8, 2020, and entitled “Engineered pH-dependent anti-CD3 antibodies and methods for their production and use”.

[0002] Related Applications

[0003] This application claims priority to U.S. Provisional Application No. 62 / 858,968, filed on June 7, 2019, the contents of which are incorporated by reference in their entirety.

[0004] Sequence Listing

[0005] This application contains a sequence listing, which has been submitted electronically in ASCII format and 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. Technical Field

[0006] The present invention particularly relates to engineered pH-dependent anti-cluster of differentiation 3 (CD3) antibodies (including multispecific antibodies and functional fragments thereof), and methods and reagents for the identification, isolation, preparation and use thereof. Background Art

[0007] Cell proliferative disorders such as cancer are characterized by the uncontrolled growth of a subpopulation of cells. They are the leading cause of death in developed countries and the second leading cause of death in developing countries, with the total number of new cancer cases expected to increase to 23.6 million per year by 2030. The National Cancer Institute estimates that nearly 2 million new cancer cases will be diagnosed in the United States and more than 600,000 Americans will die from cancer in 2018. Therefore, cancer care represents a significant and increasing burden on society.

[0008] The idea of ​​using the cytotoxic capacity of T cells to kill tumor cells by using CD3 targeting bispecific antibodies dates back to the mid-1980s. (Staerz et al. Nature 1985314:628-32). Many bispecific antibodies developed so far contain a first binding site specific for CD3 for T cell recruitment and activation, and a second binding site for a targeted disease-related antigen (such as an antigen produced by a tumor cell). CD3 bispecific antibodies trigger the CD3 surface receptor on T cells by binding to its second target protein expressed on the tumor, so that available T cells can bind to the targeted expression cells through the bridging of CD3 bispecific antibodies, regardless of the peptide / MHC specificity of its T cell receptor. (See, for example, Bassan, 2012, Blood 120:5094-95). Bridging T cells and tumor cells using CD3 bispecific antibodies can induce significant regression of advanced malignancies, and in some cases lead to complete remission. Currently, more than 25 different CD3 bispecific antibodies are in clinical development for the treatment of hematological malignancies or solid cancers by targeting CD19, CD20, CD33 and CD123, or EpCAM, HER2, PSMA and CEA, respectively (see, e.g., Liu et al. Front Immunol 2017 8:38).

[0009] Although bispecific antibodies have shown significant benefits over monospecific antibodies for the treatment and detection of cancer, the widespread commercial application of bispecific antibodies is hindered by the lack of effective / low-cost production methods, the lack of stability of bispecific polypeptides, and the lack of long half-life in humans. In the past few decades, various methods for producing bispecific monoclonal antibodies have been developed. However, many candidate bispecific antibodies with fine selectivity and high efficacy for the target of interest often have problems in downstream development and clinical efficacy activity, including multi-specific binding (or "multi-specificity"); off-target binding; non-specific binding; poor expression levels or expression profiles in eukaryotic host cells (such as mammalian host cells and yeast cells); poor chemical and physical properties, such as poor stability during storage (e.g., poor / low "shelf life" stability), poor (low) solubility, poor (high) viscosity, aggregation tendency, etc.; and poor clinical and biophysiological properties, such as poor pharmacokinetic properties, poor pharmacodynamic properties, fast or poor in vivo clearance, short circulation half-life, some of which lead to the termination of their development.

[0010] There are certain techniques and assays for evaluating many of the above-mentioned developability characteristics of discovered antibodies ("post-discovery antibodies") in the context of downstream development activities, such as CIC, SIC, BVP-ELISA, TMA and other assays; however, such assays are generally not suitable for high-throughput formats in early antibody discovery platforms. In addition, the evaluation of these properties typically requires milligram to gram quantities of protein, thus often placing a de facto limit on the number of leads that can be realistically considered for development, and thus reducing the likelihood of project success. As a result, a large amount of resources are often expended trying to repair poorly performing lead candidates that have little available backup in the late stages of development.

[0011] A variety of anti-CD3 antibodies are known in the art, including monoclonal antibodies and bispecific antibody forms. See, for example, U.S. Patent Nos. 7,262,276; 7,635,472; 7,862,813; 9,587,021; and 10,174,124. However, many of these anti-CD3 antibodies have developability issues (such as those outlined above), and / or induce the production of cytokines, generally leading to toxic cytokine release syndrome (CRS). Because the anti-CD3 binding domains of bispecific antibodies engage all T cells, CD4 T cell subsets with high cytokine production are recruited. Therefore, there is an unmet need for providing anti-CD3 antibodies, which show a desired developability and / or CRS risk profile and are, for example, specifically combined with CD3 expressed on T cells, activated T cells and (re) directed activated T cells to kill target cells and act in a manner that reduces the risk of inducing CRS. It is safe and effective.

[0012] One method for developing a CD3 binding domain that exhibits a desired CRS risk profile is to engineer a CD3 binding domain with pH-dependent antigen binding. Histidine and / or other ionizable residues incorporated into the binding interface of antibodies and other proteins have previously been used for engineering pH-dependent antigen binding (see, e.g., 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 that result in pH-dependent differences in binding affinity (Gera et al., PLOS ONE 7 (11) e48928.doi: 10.1371 / 2012). Recognizing that the pH range of human blood is about 7.6-7.8, yet tumor cells have an extracellular pH of about 6.3-6.5 due, at least in part, to the accumulation of metabolic acids that cannot be effectively cleared due to poor tumor angiogenesis, Applicants' engineered pH-dependent CD3 binding domains with preferential CD3 binding at low (lower) pH values ​​promote binding and activity in and around the tumor microenvironment. Without being bound by theory, it is believed that CD3 binding domains engineered to preferentially bind CD3 at lower pH (e.g., pH ~6) can produce selective and sustained cytotoxic activity at or around tumor sites, thereby potentially reducing or eliminating off-target effects and improving half-life and dosage. Summary of the invention

[0013] The present disclosure relates to engineered pH-dependent anti-CD3 antibodies and antigen-binding fragments thereof, which optionally bind to CD3 and / or cells expressing CD3 with a higher binding affinity at pH 6.0 than at physiological pH (pH 7.4); and methods of using the engineered pH-dependent anti-CD3 antibodies and antigen-binding fragments thereof.

[0014] In certain embodiments, the disclosure provides antibodies 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.

[0015] In certain embodiments, the present disclosure provides antibodies comprising a CD3 binding domain selected from the group consisting of ADI-48592, ADI-48595, ADI-48650, ADI-48652, ADI-48662, and ADI-48666.

[0016] In certain embodiments, the present disclosure provides antibodies 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.

[0017] Analysis of 258 unique clones identified using the methods described herein revealed a consensus motif within the CDRH3 region. In some embodiments, the disclosure provides an antibody comprising a CD3 binding domain CDRH3, wherein the CDRH3 binding domain comprises a consensus motif comprising the sequence AX1DX2YX3HX4FYDV, wherein X1 is R or H, wherein X2 is A or H, wherein X3 is G, H or P, wherein X4 is Y, H, D, V, E, S, N, L, M, I, G, A, Q or T, and wherein, optionally, at least one of X1, X2, X3 and X4 is replaced by H (SEQ ID NO: 1).

[0018] In some embodiments, the present disclosure provides an antibody comprising a CD3 binding domain CDRH3, wherein the CDRH3 binding domain has a consensus motif represented by the sequence ARDX1YGX2X3X4YDX5, wherein X1 is A or H, wherein X2 is R or H, wherein X3 is H or Y, wherein X4 is F or H, wherein X5 is H or V, and wherein, optionally, at least one of X1, X2, X3, X4 and X5 is substituted with H (SEQ ID NO: 2).

[0019] In some embodiments, the present disclosure provides an antibody comprising a CD3 binding domain CDRH3, wherein the CDRH3 binding domain comprises a consensus motif comprising the sequence ARDAHX1X2YX3X4DX5, wherein X1 is G, E or R, wherein X2 is R or H, wherein X3 is F or H, wherein X4 is Y or H, wherein X5 is V or H, and wherein, optionally, at least one of X2, X3, X4 and X5 is replaced by H (SEQ ID NO: 3).

[0020] In some embodiments, the present disclosure provides an antibody comprising a CD3 binding domain CDRH3, wherein the CDRH3 binding domain comprises a consensus motif comprising the sequence ARDAX1HRX2FYDV, wherein X1 is H, Y, S, G, A, T, V or R, wherein X2 is Y or H, and wherein, optionally, at least one of X1 and X2 is substituted with H (SEQ ID NO: 4).

[0021] In some embodiments, the present disclosure provides an antibody comprising a CD3 binding domain CDRH3, wherein the CDRH3 binding domain comprises a consensus motif comprising the sequence ARDX1YHRYFYDX2, wherein X1 is H or A, wherein X2 is H, V or M, and wherein, optionally, at least one of X1 and X2 is substituted with H (SEQ ID NO: 5).

[0022] In some embodiments, the present disclosure provides an antibody comprising a CD3 binding domain CDRH3, wherein the CDRH3 binding domain comprises a consensus motif comprising the sequence AX1DAYX2X3X4HX5DV, wherein X1 is R or H, wherein X2 is G or H, wherein X3 is H or R, wherein X4 is N, F or Y, wherein X5 is Y or H, and wherein, optionally, at least one of X1, X2, X3 and X5 is replaced by H (SEQ ID NO:6).

[0023] In some embodiments, the present disclosure provides an antibody comprising a CD3 binding domain CDRH3, wherein the CDRH3 binding domain comprises a consensus motif comprising the sequence ARDX1X2GRYFYDV, wherein X1 is M, Q or H, wherein X2 is R or H, and wherein, optionally, at least one of X1 and X2 is replaced by H (SEQ ID NO: 7).

[0024] In some embodiments, the present disclosure provides an antibody comprising a CD3 binding domain CDRH3, wherein the CDRH3 binding domain comprises a consensus motif comprising the sequence ARDX1X2X3RYFYDX4, wherein X1 is H or A, wherein X2 is T, Y or H, wherein X3 is G or H, wherein X4 is V or H, and wherein, optionally, at least one of X1, X2, X3 and X4 is substituted with H (SEQ ID NO: 8).

[0025] In some embodiments, the present disclosure provides an antibody comprising a CD3 binding domain, CDRH3, wherein the CDRH3 binding domain comprises a consensus motif comprising the sequence AX1DX2XXXXXXXXXXXXDX9, wherein X1 is R or H, wherein X2 is A, H, M or Q, wherein X3 is Y, H, S, G, A, T, V or R; wherein X4 is G, H, P, E or R; wherein X5 is H or R, wherein X6 is Y, N, F, H, D, E, S, L, M, I, G, A, Q or T; wherein X7 is F or H; wherein X8 is Y or H; wherein X9 is V, H or M; and optionally, wherein at least one of X1, X2, X3, X4, X5, X6, X7, X8 and X9 is H (SEQ ID NO: 58).

[0026] In some embodiments, the present disclosure provides an antibody comprising a CD3 binding domain CDRH3, wherein the CDRH3 binding domain comprises a consensus motif comprising the sequence ARDAX1XXXFYDX5, wherein X1 is T, H or Y, wherein X2 is G or H, wherein X3 is H or R, wherein X4 is V or Y, wherein X5 is V or H, and wherein, optionally, at least one of X1, X2, X3 and X5 is substituted with H (SEQ ID NO: 593).

[0027] In some embodiments, the present disclosure provides an antibody comprising a CD3 binding domain CDRH3, wherein the CDRH3 binding domain comprises a consensus motif comprising the sequence AX1DX2XXXX5XXXX7YDX8, wherein X1 is R or H, wherein X2 is H or A, wherein X3 is H or Y, wherein X4 is H, G or P, wherein X5 is R or H, wherein X6 is Y, I or V, wherein X7 is F or H, wherein X8 is V or H, and wherein, optionally, at least one of X1, X2, X3, X4, X5, X7 and X8 is replaced by H (SEQ ID NO: 596).

[0028] In some embodiments, the present disclosure provides an antibody comprising a CD3 binding domain CDRH2, wherein the CDRH2 binding domain comprises a consensus motif comprising the sequence WIDLENANTIYDAKFQG (SEQ ID NO: 9).

[0029] In some embodiments, the present disclosure provides an antibody comprising a CD3 binding domain CDRH2, wherein the CDRH2 binding domain comprises a consensus motif comprising the sequence X1INPX2TGX3TX4YSQKFQG, wherein X1 is W or Y, wherein X2 is A, S, D, G, N, L, V, H or Q, wherein X3 is A, T or S, and wherein X4 is K, V, T, D, Y, F or A (SEQ ID NO: 10).

[0030] In some embodiments, the present disclosure provides an antibody comprising a CD3 binding domain CDRH2, wherein the CDRH2 binding domain comprises a consensus motif comprising the sequence X1IX2AGTGX3TX4YSQKFQG, wherein X1 is W, Y or F, wherein X2 is T, N or D, wherein X3 is A, T or L, and wherein X4 is A, K, V, H, T or N (SEQ ID NO: 11).

[0031] In some embodiments, the present disclosure provides an antibody comprising a CD3 binding domain CDRH2, wherein the CDRH2 binding domain comprises a consensus motif comprising the sequence X1IDAGTGX2TX3YSQKFQG, wherein X1 is S or W, wherein X2 is L, N, D or F, and wherein X3 is D, Y or K (SEQ ID NO: 12).

[0032] In some embodiments, the present disclosure provides an antibody comprising a CD3 binding domain CDRH2, wherein the CDRH2 binding domain comprises a consensus motif comprising the sequence X1IX2AGTGATX3YSQKFQG, wherein X1 is G, D or S, wherein X2 is I or D, and wherein X3 is K or D (SEQ ID NO: 13).

[0033] In some embodiments, the present disclosure provides an antibody comprising a CD3 binding domain, wherein the CDRH2 binding domain comprises a consensus motif comprising the sequence WINPX1TGNTX2YSQKFQG, wherein X1 is D, T, L, S or A, and wherein X2 is D, V, L or N (SEQ ID NO: 14).

[0034] In some embodiments, the present disclosure provides an antibody comprising a CD3 binding domain CDRH2, wherein the CDRH2 binding domain comprises a consensus motif comprising the sequence X1INAGTGX2TX3YSQKFQG, wherein X1 is Y or W, wherein X2 is N, D or A, and wherein X3 is I or V (SEQ ID NO: 15).

[0035] In some embodiments, the present disclosure provides an antibody comprising a CD3 binding domain CDRH2, wherein the CDRH2 binding domain comprises a consensus motif comprising the sequence X1INPX2TGX3TKYSQKFQG, wherein X1 is W or Y, wherein X2 is D, I or Y, and wherein X3 is D, Y or E (SEQ ID NO: 16).

[0036] In some embodiments, the present disclosure provides an antibody comprising a CD3 binding domain CDRH2, wherein the CDRH2 binding domain comprises a consensus motif comprising the sequence SIX1AGTGX2TKYSQKFQG, wherein X1 is N or V, and wherein X2 is A or I (SEQ ID NO: 17).

[0037] In some embodiments, the present disclosure provides an antibody comprising a CD3 binding domain CDRH2, wherein the CDRH2 binding domain comprises a consensus motif comprising the sequence SINAGTGX1TX2YSQKFQG, wherein X1 is F or N, and wherein X2 is Y or D (SEQ ID NO: 18).

[0038] In some embodiments, the present disclosure provides an antibody comprising a CD3 binding domain CDRH2, wherein the CDRH2 binding domain comprises a consensus motif comprising the sequence X1IX2X3GTGX4TDYSQKFQG, wherein X1 is D or W, wherein X2 is N or H, wherein X3 is A or S, and wherein X4 is A or N (SEQ ID NO: 19).

[0039] In some embodiments, the present disclosure provides an antibody comprising a CD3 binding domain CDRH2, wherein the CDRH2 binding domain comprises a consensus motif comprising the sequence: WIDPX1TGATX2YSQKFQG, wherein X1 is N, H or Y, and wherein X2 is V or K (SEQ ID NO: 20).

[0040] In some embodiments, the present disclosure provides an antibody comprising a CD3 binding domain CDRH2, wherein the CDRH2 binding domain comprises a consensus motif comprising the sequence WIX1PX2TGNTKYSQKFQG, wherein X1 is D or N, and wherein X2 is L, I or V (SEQ ID NO: 21).

[0041] In some embodiments, the present disclosure provides an antibody comprising a CD3 binding domain CDRH2, wherein the CDRH2 binding domain comprises a consensus motif comprising the sequence SINAGDANTKYSQKFQG (SEQ ID NO: 22).

[0042] In some embodiments, the present disclosure provides an antibody comprising a CD3 binding domain CDRH2, wherein the CDRH2 binding domain comprises a consensus motif comprising the sequence X1IDPX2TGATX3YSQKFQG, wherein X1 is D or W, wherein X2 is D or V, and wherein X3 is E or D (SEQ ID NO: 23).

[0043] In some embodiments, the present disclosure provides an antibody comprising a CD3 binding domain CDRH2, wherein the CDRH2 binding domain comprises a consensus motif comprising the sequence WINAGDAATVYSQKFQG (SEQ ID NO: 24).

[0044] In some embodiments, the present disclosure provides an antibody comprising a CD3 binding domain, CDRH2, wherein the CDRH2 binding domain comprises a consensus motif comprising the sequence XI IX2XXXXXXTX8YSQKFQG, wherein X1 is W, S, Y, F, G or D, wherein X2 is N, T, D, V or H, wherein X3 is A, P or S, wherein X4 is G, A, S, N, D, L, V, H, Q, T, I or Y, wherein X5 is D or T, wherein X6 is A or G, wherein X7 is A, N, T, S, L, D, F, Y or E, wherein X8 is V, K, T, D, Y, F, A, H, N, L, I or E, and optionally, wherein at least one of X1, X2, X3, X4, X5, X6, X7 and X8 is H (SEQ ID NO: 59).

[0045] In some embodiments, the present disclosure provides an antibody comprising a CD3 binding domain CDRH2, wherein the CDRH2 binding domain comprises a consensus motif comprising the sequence WIDAGTGX1TX2YSQKFQG, wherein X1 is L, F, N or A, and wherein X2 is T or K (SEQ ID NO: 595).

[0046] In some embodiments, the present disclosure provides an antibody comprising a CD3 binding domain CDRH1, wherein the CDRH1 binding domain comprises FNIKDYHMH (SEQ ID NO: 25), SNIKDYYMH (SEQ ID NO: 26), or SNIKDYHMH (SEQ ID NO: 27).

[0047] In some embodiments, the present disclosure provides an antibody comprising a CD3 binding domain CDRH1, wherein the CDRH1 binding domain comprises a consensus motif comprising the sequence YTFX1XXXMH, wherein X1 is A, K, D, Q, E, N, T, L, Y, S, P, G, H or V, wherein X2 is T, S or A, wherein X3 is Y or I, and wherein X4 is A, D, N, S, Y, T, I, V, L, E, P, R or G (SEQ ID NO:28).

[0048] In some embodiments, the present disclosure provides an antibody comprising a CD3 binding domain CDRH1, wherein the CDRH1 binding domain comprises a consensus motif comprising the sequence YTFX1X2X3X4MH, wherein X1 is T, D, A, N or V, wherein X2 is D, E, G or Q, wherein X3 is Y or D, and wherein X4 is D, A, E, N, S, Y or V (SEQ ID NO: 29).

[0049] In some embodiments, the present disclosure provides an antibody comprising a CD3 binding domain CDRH1, wherein the CDRH1 binding domain comprises a consensus motif comprising the sequence YTFTSX1X2MH, wherein X1 is A, D or T, and wherein X2 is D, F, A, M, V or Y (SEQ ID NO: 30).

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

[0051] In some embodiments, the present disclosure provides an antibody comprising a CD3 binding domain CDRH1, wherein the CDRH1 binding domain comprises a consensus motif comprising the sequence YTFX1X2YVMH, wherein X1 is I or N, and wherein X2 is K or R (SEQ ID NO: 32).

[0052] In some embodiments, the present disclosure provides an antibody comprising a CD3 binding domain CDRH1, wherein the CDRH1 binding domain comprises a consensus motif comprising the sequence FNIKDYYMH (SEQ ID NO: 47).

[0053] In some embodiments, the present disclosure provides an antibody comprising a CD3 binding domain CDRH1, wherein the CDRH1 binding domain comprises a consensus motif comprising the sequence YTFX1X2YX3MH, wherein X1 is E, S or T, wherein X2 is S or D, and wherein X3 is A or D (SEQ ID NO: 31).

[0054] In some embodiments, the present disclosure provides an antibody comprising a CD3 binding domain CDRL3, wherein the CDRL3 binding domain comprises a consensus motif comprising the sequence XXXXRX6, wherein X1 is H, K or G, wherein X2 is Q or H, wherein X3 is Y or H, wherein X4 is S, H, D, T, V, M or L, wherein X5 is R or H, wherein X6 is T or H, and wherein, optionally, at least one of X1, X2, X3, X4, X5 and X6 is replaced by H (SEQ ID NO: 33).

[0055] In some embodiments, the present disclosure provides an antibody comprising a CD3 binding domain, CDRL3, wherein the CDRL3 binding domain comprises a consensus motif comprising the sequence KQSYX1X2RT, wherein X1 is H, V, K, W, R, L, G, Y or Q, wherein X2 is H, L, E, W, G, M, P, T, Q or V, and wherein, optionally, at least one of X1 and X2 is replaced by H (SEQ ID NO: 34).

[0056] In some embodiments, the present disclosure provides an antibody comprising a CD3 binding domain CDRL3, wherein the CDRL3 binding domain comprises a consensus motif comprising the sequence X1QSX2HX3RT, wherein X1 is K or H, wherein X2 is H, Y, M, S, L, E, G or W, wherein X3 is R or K, and wherein, optionally, at least one of X1 and X2 is replaced by H (SEQ ID NO: 35).

[0057] In some embodiments, the present disclosure provides an antibody comprising a CD3 binding domain CDRL3, wherein the CDRL3 binding domain comprises a consensus motif comprising the sequence KQSX1X2X3RT, wherein X1 is Y or H, X2 is T, S, V or K, X3 is R or H, and wherein, optionally, at least one of X1 and X3 is replaced by H (SEQ ID NO: 36).

[0058] In some embodiments, the present disclosure provides an antibody comprising a CD3 binding domain CDRL3, wherein the CDRL3 binding domain comprises a consensus motif comprising the sequence KQSX1X2X3RT, wherein X1 is H or Y, wherein X2 is T, S or Q, wherein X3 is R or H, and wherein, optionally, at least one of X1 and X3 is replaced by H (SEQ ID NO: 36).

[0059] In some embodiments, the present disclosure provides an antibody comprising a CD3 binding domain, CDRL3, wherein the CDRL3 binding domain comprises a consensus motif comprising the sequence X1QSX2XXXRT, wherein X1 is K or H, wherein X2 is Y or H, wherein X3 is S, H, L, V or K, wherein X4 is H, R or E, and wherein, optionally, at least one of X1, X2, X3 and X4 is replaced by H (SEQ ID NO: 598).

[0060] In some embodiments, the present disclosure provides an antibody comprising a CD3 binding domain CDRL2, wherein the CDRL2 binding domain comprises a consensus motif comprising the sequence: WASTRES (SEQ ID NO: 37).

[0061] In some embodiments, the present disclosure provides an antibody comprising a CD3 binding domain CDRL1, wherein the CDRL1 binding domain comprises a consensus motif comprising the sequence KSSQSLLX1X2X3X4GX5NX6LA, wherein X1 is N or H, wherein X2 is A, R or T, wherein X3 is R or H, wherein X4 is T, P or E, wherein X5 is H or K, wherein X6 is H or Y, and wherein, optionally, at least one of X1, X3, X5 and X6 is replaced by H (SEQ ID NO: 38).

[0062] In some embodiments, the present disclosure provides an antibody comprising a CD3 binding domain CDRL1, wherein the CDRL1 binding domain comprises a consensus motif comprising the sequence KSSQSLLX1AX2THX3NX4LA, wherein X1 is N or H, wherein X2 is R or H, wherein X3 is K or H, wherein X4 is Y or H, and wherein, optionally, at least one of X1, X2, X3 and X4 is replaced by H (SEQ ID NO: 39).

[0063] In some embodiments, the present disclosure provides an antibody comprising a CD3 binding domain CDRL1, wherein the CDRL1 binding domain comprises a consensus motif comprising the sequence KSSQSLLNASTAKNYLA (SEQ ID NO: 40) or KSSQSLLNARTRTNYLA (SEQ ID NO: 41).

[0064] In some embodiments, the present disclosure provides an antibody comprising a CD3 binding domain CDRL1, wherein the CDRL1 binding domain comprises a consensus motif comprising the sequence KSSQSLLNX1X2X3GX4NX5LA, wherein X1 is S or A, wherein X2 is R or H, wherein X3 is E or T, wherein X4 is H or K, wherein X5 is H or Y, and wherein, optionally, at least one of X2, X4 and X5 is replaced by H (SEQ ID NO:42).

[0065] In some embodiments, the present disclosure provides an antibody comprising a CD3 binding domain CDRL1, wherein the CDRL1 binding domain comprises a consensus motif comprising the sequence KSSQSLLNX1X2TGX3NYLA, wherein X1 is A or S, wherein X2 is R or H, wherein X3 is H or K, and optionally, wherein at least one of X2 and X3 is replaced by H (SEQ ID NO: 594).

[0066] In some embodiments, the present disclosure provides an antibody comprising a CD3 binding domain CDRL1, wherein the CDRL1 binding domain comprises a consensus motif comprising the sequence KSSQSLLX1AX2X3X4X5NX6LA, wherein X1 is N or H, wherein X2 is R or H, wherein X3 is T or E, wherein X4 is G or H, wherein X5 is H or K, wherein X6 is H or Y, and wherein, optionally, at least one of X1, X2, X4, X5 and X6 is replaced by H (SEQ ID NO: 597).

[0067] In some embodiments, the present disclosure provides antibodies or antigen-binding fragments comprising a CDRH3 binding domain comprising a consensus motif comprising the sequence ARDAX1X2X3X4FYDX5, wherein X1 is T, H or Y, wherein X2 is G or H, wherein X3 is H or R, wherein X4 is V or Y, wherein X5 is V or H, and wherein, optionally, at least one of X1, X2, X3 and X5 is H (SEQ ID NO: 593); a CDRH2 binding domain comprising a consensus motif comprising the sequence WIDLENANTIYDAKFQG (SEQ ID NO: 9); a CDRH1 binding domain comprising a consensus motif comprising the sequence FNIKDYYMH (SEQ ID NO:47); a CDRL3 binding domain comprising a consensus motif comprising the sequence KQSX1X2X3RT, wherein X1 is H or Y, wherein X2 is T, S or Q, wherein X3 is R or H, and optionally, wherein at least one of X1 and X3 is H (SEQ ID NO:36); a CDRL2 binding domain comprising a consensus motif comprising the sequence WASTRES (SEQ ID NO:37); and / or a CDRL1 binding domain comprising a consensus motif comprising the sequence KSSQSLLNX1X2TGX3NYLA, wherein X1 is A or S, wherein X2 is R or H, wherein X3 is H or K, and optionally, wherein 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 binding agent comprising a CD3 binding domain selected from ADI-48592, ADI-48595, ADI-48650, ADI-48652, ADI-48662, and ADI-48666.

[0068] In some embodiments, the present disclosure provides antibodies or antigen-binding fragments comprising a CDRH3 binding domain comprising a consensus motif comprising the sequence AX1DX2XXXX5X6X7YDX8, wherein X1 is R or H, wherein X2 is H or A, wherein X3 is H or Y, wherein X4 is H, G or P, wherein X5 is R or H, wherein X6 is Y, I or V, wherein X7 is F or H, wherein X8 is V or H, and optionally, wherein 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 comprising the sequence WIDLENANTIYDAKFQG (SEQ ID NO: 9) or the sequence WIDAGTGX1TX2YSQKFQG, wherein X1 is L, F, N or A, and wherein X2 is T or K (SEQ ID NO: 103); NO:595); a CDRH1 binding domain comprising a consensus motif comprising the sequence FNIKDYYMH (SEQ ID NO:47) or the sequence YTFX1X2YX3MH, wherein X1 is E, S or T, wherein X2 is S or D, and wherein X3 is A or D (SEQ ID NO:31); a CDRL3 binding domain comprising a consensus motif comprising the sequence X1QSX2X3X4RT, wherein X1 is K or H, wherein X2 is Y or H, wherein X3 is S, H, L, V or K, wherein X4 is H, R or E, and optionally, wherein at least one of X1, X2, X3 and X4 is H (SEQ ID NO:598); a CDRL2 binding domain comprising a consensus motif comprising the sequence WASTRES (SEQ ID NO:37); and / or a CDRL1 binding domain containing a consensus motif comprising the sequence KSSQSLLX1AX2X3X4X5NX6LA, wherein X1 is N or H, wherein X2 is R or H, wherein X3 is T or E, wherein X4 is G or H, wherein X5 is H or K, wherein X6 is H or Y, and wherein, optionally, at least one of X1, X2, X4, X5 and X6 is H (SEQ ID NO:597).In some embodiments, the antibody or antigen-binding fragment is designated as a Group 2 binding agent 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.

[0069] In some embodiments, the present disclosure provides an anti-CD3 antibody or antigen-binding fragment as described herein, wherein the antibody or antigen-binding fragment can elicit T cell activation or T cell killing while exhibiting a reduced propensity to elicit cytokine production to levels capable of inducing cytokine release syndrome.

[0070] In some embodiments, the present disclosure provides an anti-CD3 antibody or antigen-binding fragment as described herein, wherein the antibody or antigen-binding fragment may comprise a multispecific antibody.

[0071] In some embodiments, the present disclosure provides an anti-CD3 antibody or antigen-binding fragment described herein, wherein the antibody or antigen-binding fragment may comprise a bispecific antibody.

[0072] In some embodiments, the present disclosure provides an anti-CD3 antibody or antigen-binding fragment described herein, wherein the antibody or antigen-binding fragment may comprise a scFv.

[0073] In some embodiments, the present disclosure provides an anti-CD3 antibody or antigen-binding fragment described herein, wherein the antibody or antigen-binding fragment may comprise at least a second antigen-binding domain that specifically binds to: an oncology 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.

[0074] In some embodiments, the present disclosure provides an anti-CD3 antibody or antigen-binding fragment as described herein, wherein the antibody or antigen-binding fragment may comprise at least a second antigen-binding domain that specifically binds to an antigen selected from the group consisting of: 17-IA, 4-1BB, 4Dc, 6-keto-PGF1a, 8-iso-PGF2a, 8-oxo-dG, Al adenosine receptor, A33, ACE, ACE-2, activin, activin A, activin AB, activin B, activin C, activin RIA, activin RIA ALK-2, activin RIB ALK-4, activin RIIA, activin RUB, ADAM, ADAM10, ADAM12, ADAM 15, ADAM 17 / T ACE, ADAM8, ADAM9, ADAMTS, ADAMTS4, ADAMTS5, addressin, aFGF, ALCAM, ALK, ALK-1, ALK-7, alpha-l-antitrypsin, alpha-V / beta-1 antagonist, ANG, Ang, APAF-1, APE, APJ, APP, APRIL, AR, ARC, ART, artesunate (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-2BMP-2a, BMP-3osteomorphin, BMP-4BM P-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), cancer-associated antigen, cathepsin A, cathepsin B, cathepsin C / DPPI, cathepsin D, cathepsin E, cathepsin H, cathepsin L, cathepsin O, cathepsin S, cathepsin V, cathepsin X / Z / P, CBL, CCI, CCK2, CCL, CCL1, CCL11, CCL12, CCL13, CCL 14, CCL15, CCL16, CCL17. CCL18, CCL19, CCL2, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9 / 10, CCR, CCR1, CCR10, CCR10, CC R2, 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, C D23, 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, Clostridium botulinum toxin, Clostridium perfringens 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, CXC L4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCR, CXCR1, CX CR2, CXCR3, CXCR4, CXCR5, CXCR6, cytokeratin tumor-associated antigen, DAN, DCC, DcR3, DC-SIGN, decay-accelerating factor, des(l-3)-IGF-I (brain IGF-1), Dhh, digoxin, DNAM-1, Dnase, Dpp, DPPIV / CD26, Dtk, ECAD, EDA, EDA-A1, EDA-A2, EDAR, EGF, EGFR (ErbB-1), EMA, EMMPRIN, 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, fibrinogen, 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-α1, GFR-α2, GFR-α3, 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 gB envelope glycoprotein, HCMV) gH envelope glycoprotein, HCMV UL, hematopoietic 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, HIVIIIB gp 120V3 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-18 R, IL-23, interferon (INF)-α, INF-β, INF-γ, inhibin, iNOS, insulin A chain, insulin B chain, insulin-like growth factor 1, integrin α2, integrin α3, integrin α4, integrin α4 / βl, integrin, α4 / β7, integrin α5 (αV), integrin α5 / βl, integrin α5 / β3, integrin α6, integrin βl, integrin β2, interferon γ, IP-10, 1-TAC, JE, kallikrein 2, kallikrein 5, kallikrein 6, kallikrein 11, kallikrein 12, kallikrein 14, kallikrein 15, kallikrein LI, kallikrein L2, kallikrein Kallikrein L3, Kallikrein L4, KC, KDR, Keratinocyte Growth Factor (KGF), Laminin 5, LAMP, LAP, LAP (TGF-1), Potential TGF-1, Potential TGF-1bpl, LBP, LDGF, LECT2, Lefty, Lewis-Y Antigen, Lewis-Y Related Antigen, LFA-1, LFA-3, Lfo, LIF, LIGHT, Lipoprotein, LIX, LKN, Lptn, L-Selectin, LT-a, LT-b, LTB4, LTBP-1, Pulmonary Surfactant, Luteinizing Hormone, Lymphotoxin B Receptor, Mac-1, MAdCAM, MAG, MAP2, MARC, MCAM, MCAM, MCK-2, MCP, M-CSF, MDC, Mer, α-metalloproteinase, MGDF receptor, MGMT, MHC (HLA-DR), MIF, MIG, MIP, MIP-1-α, 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, Mullerian-inhibiting substance (MUC18),substance), Mug, MuSK, NAIP, NAP, NCAD, N-cadherin, NCA 90, NCAM, NCAM, Neprilysin, neurotrophin-3, -4 or -6, neurotrophin (Neurturin), neuron growth factor (NGF), NGFR, NGF-β, nNOS, NO, NOS, Npn, NRG-3, NT, NTN, OB, OGG1, OPG, OPN, OSM, OX40L, OX40R, pl50, p95, PADPr, parathyroid hormone, PARC, PARP, PBR, PBSF, PCAD, P-cadherin, PCNA, PDGF, PDGF, PDK-1, PECAM, PEM, PF4, PGE, PGF, PGI2, PGJ2, PIN, PLA2, placental alkaline phosphatase (PLAP), PIGF, PLP, PP14, proinsulin, prorelaxin, protein C, PS, PSA, PSCA, prostate-specific membrane antigen (PSMA), PTEN, PTHrp, Ptk, PTN, R51, RANK, RANKL, RANTES, relaxin A chain, relaxin B chain, renin, respiratory syncytial virus (RSV) F, 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 receptors (e.g., T cell receptor α / β), TdT, TECK, TEM1, TEM5, TEM7, TEM8, TERT, testis P LAP-like alkaline phosphatase, TfR, TGF, TGF-α, TGF-β, TGF-βPan specific, TGF-βRI (ALK-5), TGF-βRII, TGF-βRllb, TGF-βRIII, TGF-βl, TGF-β2, TGF-β3, TGF-β4, TGF-β5, thrombin, thymus Ck-1, thyroid stimulating hormone, Tie, TIMP, TIQ, tissue factor, TMEFF2, Tmpo, TMPRSS2, TNF, TNF-α, TNF-αβ, TNF-β2, TNFc, TNF-RI, TNF-RII, TNFRSF10A (TRAIL Rl Apo-2, DR4), TNFRSFIOB (TRAIL R2DR5, KILLER, TRICK-2A, TRICK-B), TNFRSF10C (TRAILR3DcRl、LIT、TRID)、TNFRSF10D(TRAIL R4DcR2、TRUNDD)、TNFRSF11A(RANK ODF R、TRANCE R)、TNFRSFllB(OPG OCIF、TR1)、TNFRSF12(TWEAK R). FN14)、TNFRSF13B(TACI)、TNFRSF13C(BAFF R)、TNFRSF14(WHEM ATAR、HveA、LIGHT R、TR2)、TNFRSF16(NGFR p75NTR)、TNFRSF17(BCMA)、TNFRSF 18(GITR). AITR)、TNFRSF19(TROY CROWN、TRADE)、TNFRSF19L(RELT)、TNFRSFIA(TNF RI CD120a、p55-60)、TNFRSFIB(TNF RIICD120b, p75-80, TNFRSF26(TNFRH3), TNFRSF3(LTbR TNF RIII, TNFC R), TNFRSF4(OX40 ACT35, TXGP1R), TNFRSF 5(CD40p50), TNFRSF6(Fas). Apo-1, APT1, CD95, TNFRSF6B(DcR3M68, TR6), TNFRSF7(CD27), TNFRSF8(CD30), TNFRSF9(4-1BB). CD137, ILA, TNFRSF21(DR6), TNFRSF22(DcTRAILR2TNFRH2), TNFRST23(DcTRAIL Rl TNFRH1), TNFRSF25(DR3Apo-3, LARD, TR-3, TRAMP, WSL-1), TNFSF10(TRAIL). Apo-2 division, TL2, TNFSF11 (TRANCE / RANK division ODF, OPG division), TNFSF12 (TWEAK Apo-3 division, DR3 division), TNFSF13 (APRIL TALL2), TNFSF13B (BAFF). LIGHT、TALL1、THANK、TNFSF20)、TNFSF14(LIGHT LTg, TNFSF15 (TLIA / VEGI), TNFSF18, GITR, AITR, TL6, TNFSFIA, T NF-a activator (Conectin), DIF, TNFSF2), TNFSF1B (TNF-bLTa, 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, TRF, Trk, TROP-2, TSG, TSLP, tumor-associated antigen CA 125, tumor-associated antigen expressing Lewis Y related carbohydrate carbohydrate), 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, anti-von Willebrand factor (von Willebrands The main inhibitory factors include T cell death protein 1 (T-1), PD-L1 (programmed cell death ligand 1), LAG-3 (lymphocyte activation gene 3), TIM-3 (T cell immunoglobulin and mucin 3), hormone receptors and growth factors.

[0075] In some embodiments, the present disclosure provides an anti-CD3 antibody or antigen-binding fragment described herein, wherein the antibody or antigen-binding fragment may comprise 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 receptor (TLR), TLR4, TLR9, cytokines, IL-2, IL-5, IL-13, IL-6, IL-17, IL-12, IL-23, TNFa, TGFb, cytokine receptor, IL-2R, chemokine, chemokine receptor, growth factor, VEGF and HGF.

[0076] In some embodiments, the present disclosure provides an anti-CD3 antibody or antigen-binding fragment described herein, wherein the antibody or antigen-binding fragment may be contained in a chimeric antigen receptor (CAR), which optionally may contain at least one transmembrane domain, and at least one intracellular domain from a T cell receptor (optionally a CD3 zeta subunit), and at least one co-stimulatory domain.

[0077] In some embodiments, the present disclosure provides an anti-CD3 antibody or antigen-binding fragment described herein, wherein the antibody or antigen-binding fragment may comprise scFv2-Fc2 and / or scFv-IgG.

[0078] In some embodiments, the present disclosure provides an anti-CD3 antibody or antigen-binding fragment described herein, wherein the antibody or antigen-binding fragment may comprise an IgG constant domain.

[0079] In some embodiments, the disclosure provides an anti-CD3 antibody or antigen-binding fragment as described herein, wherein the antibody or antigen-binding fragment may comprise at least a second antigen-binding domain that specifically binds to an antigen, wherein the antibody comprises a multispecific format selected from the group consisting of: Fab-Fc-scFv, "bottle-opener", Mab-scFv, Mab-Fv, dual scFv, central Fv, central scFv v , single arm center scF v , Fab-Fab, Fab-F v 、mAb-F v, mAb-Fab, DART, BiTE, common light chain-IgG, TandAb, Crossover-Mab, SEED, BEAT, TrioMab and DuetMab.

[0080] In some embodiments, the disclosure provides isolated or recombinant nucleic acid sequences encoding the anti-CD3 antibodies or antigen-binding fragments described herein.

[0081] In some embodiments, the disclosure provides expression vectors comprising an isolated or recombinant nucleic acid sequence encoding an anti-CD3 antibody or antigen-binding fragment described herein.

[0082] 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 or recombinant nucleic acid sequence encoding an anti-CD3 antibody or antigen-binding fragment described herein, wherein the host cell may optionally be a mammalian cell or a yeast cell.

[0083] 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.

[0084] In some embodiments, the present disclosure provides a method for treating a disorder in a mammal in need of treatment, wherein the disorder may include a proliferative disorder, an oncological disorder, an immuno-oncological disorder, a neurological disorder, a neurodegenerative disorder, or an autoimmune disorder, and wherein the method may include administering an effective amount of at least one antibody or antigen-binding fragment described herein or a host cell (optionally an immune cell, further optionally, a T or NK cell) expressing at least one of the antibodies or antigen-binding fragments described herein. In some embodiments, the method may also include administering an additional therapeutic agent to the mammal, optionally wherein the mammal is a human.

[0085] In other embodiments, the present disclosure provides an anti-CD3 antibody or antigen-binding fragment thereof comprising one or more of CDRL1, CDRL2, and CDRL3. In some embodiments, such an antibody further comprises CDRH1, CDRH2, and CDRH3. BRIEF DESCRIPTION OF THE DRAWINGS

[0086] Figure 1A and Figure 1B Presaturation methods 1 and 2 are shown. Figure 1A: CD3 pre-saturation method #1: Yeast cells were pre-saturated with native (non-biotinylated) CD3 antigen at pH 7.4 for 10 minutes, and yeast cells were washed at pH 7.4 and incubated in pH 6.0 medium for 10 minutes to allow antigen dissociation. Control cells were washed and incubated at pH 7.4. Finally, yeast cells were incubated with biotinylated CD3 antigen (shown as star-shaped green circles) at pH 6 for 10 minutes. Control cells were incubated with biotinylated CD3 antigen at pH 7.4. The labeled binders at pH 6 were then sorted and characterized. Figure 1B : CD3 pre-saturation method #2: Yeast cells were pre-saturated with native CD3 antigen at pH 6.0 for 10 minutes, and washed at pH 6.0 and incubated at pH 7.4 or pH 6.0 for 10 minutes. Finally, yeast cells were incubated with biotinylated CD3 antigen at the opposite pH (cells incubated at pH 6.0 in the previous step were incubated at pH 7.4, whereas cells incubated at pH 7.4 in the previous step were incubated at pH 6.0) for 10 minutes. Binders labeled with biotinylated CD3 antigen were then sorted and characterized.

[0087] Figure 2 Shown are exemplary FACS graphs from rounds 1 and 2 of selection from one library. Similar binding curves were observed in all libraries. In brief, during round 1, cells were positively sorted using 100 nM human CD3 ε δ heterodimer (HuCD3-hd) at pH 6. During round 2, cells were positively sorted using 100 nM HuCD3-hd at pH 6.0, negatively sorted using 100 nM HuCD3-hd at pH 7.4, or pre-saturated using method #2 described above. Binding to cynomolgus CD3 (CyCD3-hd) at pH 6.0 was also confirmed. Arrows indicate sorted cells that advance to the next round of sorting.

[0088] Figure 3 An exemplary FACS plot from round 3 is shown and compares the input from round 2 pH 6.0 positive sort and pH 7.4 negative sort. Briefly, sorts from round 2 were incubated with 100 nM HuCD3-hd at pH 6.0 and 7.4. The overlay column shows that the input cell population (from round 2 sort) exhibited higher binding at pH 6.0 compared to pH 7.4. Presaturation / Switch Method #2 was used to advance cells to the next round of selection.

[0089] Figure 4Exemplary FACS plots from rounds 4 and 5 are shown. Round 4 compared cells incubated in 100 nM HuCD3-hd at pH 6 and pH 7.4. Round 4 also compared cells subjected to the pre-saturation / switch method at pH 6 and pH 7.4. Round 5 compared cells incubated in 100 nM HuCD3-hd or 100 nM CyCD3-hd at pH 6 (red) and pH 7.4 (grey).

[0090] Figure 5A and Figure 5B HuCD3 binding responses are shown. Figure 5A Shown are the HuCD3 binding responses at pH 6 (x-axis) compared to the HuCD3 binding responses at pH 7.4 (y-axis) for 236 unique clones from round 2 / 3 sorting output. Figure 5B Shown are KD values ​​for HuCD3 at pH 6 (x-axis) compared to HuCD3 at pH 7.4 (y-axis) for 236 unique clones from the 2 / 3 round sorting output. Blue circles represent 2 / 3 round clones obtained by pre-saturation / switch sorting at pH 6.0, yellow circles represent 2 / 3 round clones obtained by negative sorting at pH 7.4, and red circles represent parental clone ADI-26906. The results show that negative sorting at pH 7.4 in round 2 / 3 tends to produce more pH-selected binders, but with weaker pH 6.0 responses or affinities, which are designated as Group 2 binders. Positive selection and pre-saturation / switch sorting at pH 6.0 produce clones with a variety of selectivities but with higher responses / affinity. Such clones with higher affinity at pH 6.0 (e.g., K D < about 25 nM) were designated as Group 1 binders.

[0091] Figure 6 Provided are exemplary kinetics from ForteBio experiments of four clones compared to the parental clone ADI-26906. The KD for each clone was calculated at pH 7.4 and pH 6.0. The ratio KD was obtained by dividing the KD at pH 7.4 by the KD at pH 6.0. The examples demonstrate that some clones designated as Group 1 binders, such as SAD10318_P02_A05 (ADI-48595) and SAD10318_P02_C04 (ADI-48592), bind more strongly (have lower K at pH 6.0) compared to pH 7.4. DSome clones designated as Group 2 binders, such as SAD10318_P01_A03 (ADI-48587) and SAD10318_P01_E01 (ADI-48577), were non-binders at pH 7.4, but bound at pH 6.0. Figure 6 The column "Sequence" (SEQ ID NOs: 576-590, respectively, in order of appearance) highlights amino acid substitutions in the CDRH3, CDRL1, and CDRL3 regions that may contribute to differential binding. 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 relative to binding at pH 7.4), low PSR scores, and provide a range of affinities for CD3. DETAILED DESCRIPTION

[0092] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure belongs. As used herein, the term "about" when used with reference to a particular listed value means that the value may differ from the listed value by no more than 1%. For example, as used herein, the expression "about 100" includes 99 and 101 and all values ​​therebetween (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0093] It should be understood that aspects and embodiments of the present disclosure described herein include "comprising" aspects and embodiments, "consisting of" aspects and embodiments, and "consisting essentially of" aspects and embodiments.

[0094] Provided herein are anti-CD3 antibodies and antigen-binding fragments thereof that exhibit pH-dependent binding and a favorable developability profile.

[0095] "Cluster of differentiation 3" or "CD3" generally refers to any native CD3 from any vertebrate source, including mammals, such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise indicated, including, for example, CD3ε chain, CD3γ chain, CD3α chain, and CD3β chain. The term encompasses "full-length", untreated CD3 (e.g., untreated or unmodified CD3ε or CD3γ), as well as any form of CD3 produced by treatment in a cell. The term also encompasses naturally occurring variants of CD3, including, for example, splice variants or allelic variants. CD3 includes, for example, a human CD3ε protein (NCBI RefSeq No. NP-000724) having a length of 207 amino acids and a human CD3γ protein (NCBI RefSeq No. NP-000064) having a length of 182 amino acids. The term also refers to human or cynomolgus monkey CD3ε protein, SEQ ID NOs: 591 and 592, respectively (Table 4). "CD3N27" and "CD3N13" refer to the N-terminal 27 amino acids and the N-terminal 13 amino acids of CD3, respectively, and optionally contain chemical modifications or conjugations thereto.

[0096] "Anti-CD3 antibody" refers to an antibody or antigen-binding fragment thereof that is capable of binding to CD3, such as CD3ε and / or CD3γ, such as human CD3ε and / or CD3γ, with sufficient affinity and / or specificity to allow the antibody to be used as a diagnostic and / or therapeutic agent targeting CD3. In some embodiments, the anti-CD3 antibody is present at about 100 x 10 -9 M or smaller, about 50x 10 -9 M or smaller, about 25x10 -9 M or smaller, approximately 20x 10 -9 M or smaller, or about 10x 10 -9 M or less dissociation constant (K D ) binds to CD3. In some embodiments, the anti-CD3 antibody is present at about 5 x 10 -9 M or less dissociation constant (K D ) binds to CD3. In some embodiments, the anti-CD3 antibody is present at about 2.5 x 10 -9 M or less dissociation constant (K D ) binds to CD3. In some embodiments, the anti-CD3 antibody is present at about 1 x 10 -10 M or less dissociation constant (K D) binds to CD3. In some embodiments, K is measured by surface plasmon resonance (e.g., BIACORE), biolayer interferometry using, for example, a FORTEBIO Octet HTX instrument (Pall Life Sciences), or solution affinity ELISA. D In some embodiments, the KD is measured using a scFv fragment of an anti-CD3 antibody. In some embodiments, a monovalent KD is measured. In some embodiments, the anti-CD3 antibody binds to an epitope of CD3 that is conserved in CD3 from different species (e.g., human and cynomolgus monkey) that is cross-reactive.

[0097] The term "engineered pH dependency" refers to an antibody with a modified amino acid sequence that allows preferential or selective antigen binding at a certain pH. For example, a parent antibody can be engineered (e.g., by modifying the amino acid sequence) to perform pH-dependent binding. pH-dependent binding refers to the preference of an antibody to bind antigens at a given pH (or a given pH range) as compared to different pHs (or pH ranges). In one embodiment, a pH-dependent antibody preferentially or selectively binds to an antigen at a pH of about 6 as compared to a pH of about 7. The antibody sequence can be modified, for example, by replacing one or more ionizable amino acid residues such as histidine, lysine, arginine, aspartic acid, and glutamic acid. Ionizable residues can be replaced in CDR and / or FR. In some embodiments, there can be 1-10 replacements for each variant VH or VK. In some embodiments, there can be 1-6 replacements for each variant VH or VK.

[0098] The term "cytokine release syndrome" (or "CRS") refers to a pro-inflammatory positive feedback loop between cytokines and immune cells, resulting in excessive or uncontrolled pro-inflammatory cytokine release by cells within the immune system (see, e.g., Lee et al. Blood, Vol. 124, pp. 188-195 (2014) and Tisoncik et al., Microbiol Mol Biol Rev, Vol. 76, pp. 16-32 (2012)). Upon stimulation and activation, T cells release a range of cytokines to levels and degrees or varying degrees and severity that produce adverse biological / physiological effects, including acute inflammation characterized by, for example, erythema (redness), swelling or edema, burning (fever), pain (pain), and "loss of function" (loss of function). When localized in the skin or other tissues, biological / physiological effects include increased blood flow, enabling vascular leukocytes and plasma proteins to reach extravascular sites of injury, increasing local temperature and producing pain, tissue edema and extravascular pressure, and reduced tissue perfusion. Other biological / physiological effects include organ and system dysfunction, such as cardiac dysfunction, adult respiratory distress syndrome, neurotoxicity, renal and / or liver failure, and disseminated intravascular coagulation. Upon T cell stimulation, 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 implicated in the prediction and / or cause of CRS or the tendency to induce CRS.

[0099] In certain embodiments, the anti-CD3 antibodies and / or antigen-binding fragments thereof described herein are adjusted and / or modified to reduce the likelihood or severity of CRS induced by the antibody. Non-limiting exemplary modifications may include silencing the Fc region (e.g., completely removing the Fc or modifying the Fc region to reduce or eliminate effector functions), and / or masking (e.g., a polypeptide mask positioned so that it reduces or inhibits the ability of the antibody or its antigen-binding fragment to specifically bind to CD3).

[0100] The term "antibody" is used herein in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and / or antibody fragments (preferably those fragments that exhibit the desired antigen binding activity).

[0101] "Monoclonal antibody" or "mAb" refers to an antibody obtained from a substantially homogeneous antibody population, i.e., the individual antibodies constituting the population are identical and / or bind to the same epitope, except for possible variant antibodies (e.g., containing naturally occurring mutations or generated during the production of the monoclonal antibody preparation), which 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 the antigen.

[0102] Regarding multispecific antibodies, such antibodies comprise at least two different antigen-binding domains that recognize and specifically bind at least two different antigens. Regarding bispecific antibodies, such antibodies comprise two different antigen-binding domains that recognize and specifically bind at least two different antigens.

[0103] "Different antigens" may refer to different and / or distinct proteins, polypeptides, or molecules; and different and / or distinct epitopes, which may be contained within one protein, polypeptide, or other molecule.

[0104] The term "epitope" refers to an antigenic determinant that interacts with a specific antigen binding site in the variable region of an antibody molecule, called a paratope. A single antigen may have more than one epitope. Therefore, different antibodies may bind to different regions on an antigen and may have different biological effects. The term "epitope" also refers to a site on an antigen to which B cells and / or T cells respond. It also refers to an antigenic region bound by an antibody. An epitope may be defined as structural or functional. A functional epitope is typically a subset of a structural epitope and has those residues that directly contribute to the affinity of the interaction. An epitope may also be conformational, i.e., composed of nonlinear amino acids. In certain embodiments, an epitope may include a determinant grouped as a chemically active surface of a molecule such as an amino acid, a sugar side chain, a phosphoryl group, or a sulfonyl group, and in certain embodiments, may have specific three-dimensional structural features and / or specific charge characteristics.

[0105] In some cases, antibodies contain four polypeptide chains: two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0106] In other cases, the antibody may alternatively comprise a multimer thereof (e.g., IgM) or an antigen-binding fragment thereof. Each heavy chain is composed of a heavy chain variable region ("VH") and a heavy chain constant region ("CH"), which 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 region and the VL region can be further subdivided into hypervariable regions (called complementary determining regions (CDRs)), interspersed with more conservative regions (called framework regions (FRs)). Each VH and VL is composed of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In certain embodiments of the present disclosure, the FRs of the antibody (or its antigen-binding fragment) may be identical to human germline sequences, or may be naturally or artificially modified. An amino acid consensus sequence may be defined based on a side-by-side analysis of two or more CDRs. Accordingly, the CDRs in the heavy chain are designated "CDRH1," "CDRH2," and "CDRH3," and the CDRs in the light chain are designated "CDRL1," "CDRL2," and "CDRL3," respectively.

[0107] Unless specifically stated otherwise, the term "antibody" as used herein encompasses molecules comprising two immunoglobulin heavy chains and two immunoglobulin light chains (ie, a "full length antibody" or "intact antibody" or "whole antibody"), as well as antigen-binding fragments thereof.

[0108] "Antigen-binding fragment" refers to a portion of an intact antibody that binds to the antigen to which the intact antibody binds (in this case, CD3). The terms "full-length antibody," "intact antibody," and "whole antibody," etc. are used interchangeably herein and refer to an antibody that has a structure substantially similar to a native antibody.

[0109] Antigen-binding fragments of antibodies include any naturally occurring, enzymatically obtainable, synthetic or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex, including antibody fragments. Exemplary antigen-binding fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv or VH or VL domains only); and multispecific antibodies formed from antibody fragments. In some embodiments, the antigen-binding fragment of an anti-CD3 antibody described herein is a scFv.

[0110] As with full antibody molecules, antigen-binding fragments can be monospecific or multispecific (e.g., bispecific). Multispecific antigen-binding fragments of antibodies can contain at least two different variable domains, each of which is capable of specifically binding to a separate antigen or a different epitope on the same antigen. A variety of multispecific antibody formats can be used in the context of antigen-binding fragments of anti-CD3 antibodies described herein. Non-limiting examples of multispecific and bispecific formats include, e.g., Fab-Fc-scFv ("Bottle Opener") (XENCOR), Mab-scFv (XENCOR), Mab-Fv (XENCOR), Dual scFv (XENCOR), Central Fv (XENCOR), Central scFv (XENCOR), One-armed 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 (TRIONPHARMA / FRESENIUS BIOTECH), DuetMab (MEDIMMUNE), etc., as in, for example, WO95 / 09917; WO 2008 / 119566; WO 2008 / 119567; WO2011 / 121110; WO2010 / 037835; WO 2007 / 042261; WO 2007 / 110205; WO 2011 / 121110; WO 2012 / 055961; WO 2012 / 16067; WO 2016 / 086189; WO2016 / 182751; WO 2015 / 006749; WO 2014 / 049003; WO 2013 / 177101; WO 2015 / 128509;US 7,951,917; US2009 / 0252729; US2014 / 0348839; US 7,183,076; Mazor et al., Mabs, Vol. 7, pp. 377-389 (2015); Muda et al., Protein Engineering, Design, & Selection, Vol. 24, pp. 447-454 (2011); and Del Bano et al., Antibodies, Vol. 5, pp. 1-23 (2016).In some embodiments, the anti-CD3 scFv fragments described herein comprise one or more variable domains of a multispecific (eg, bispecific) antibody.

[0111] In certain embodiments, the anti-CD3 antibodies and / or antigen-binding fragments thereof as described herein are included in multispecific antibodies, particularly bispecific antibodies having binding specificity for a second antigen. Such a second antigen can be a target completely different from the first target, or a different epitope present on the same target. In some embodiments, the binding specificity is directed to two different epitopes of CD3 (e.g., CD3ε or CD3γ). In other embodiments, one of the binding specificities is directed to CD3 (e.g., CD3ε or CD3γ), while the other is directed to a different biomolecule (e.g., a cell surface antigen, such as a tumor antigen).

[0112] Non-limiting examples of second antigens directed by bispecific antibodies comprising anti-CD3 antibodies and / or antigen-binding fragments thereof as described herein include targets selected from the group consisting of 17-IA, 4-1BB, 4Dc, 6-keto-PGF1a, 8-iso-PGF2a, 8-oxo-dG, Al adenosine receptor, A33, ACE, ACE-2, activin, activin A, activin AB, activin B, activin C, activin RIA, activin RIA ALK-2, activin RIB ALK-4, activin RIIA, activin RUB, ADAM, ADAM10, ADAM12, ADAM15, ADAM17 / TACE, ADAM8, ADAM9, ADAMTS, ADAMTS4, ADAMTS5, addressin, aFGF, ALCAM, ALK, ALK-1, ALK-7, alpha-l-antitrypsin, alpha-V / beta-1 antagonist, ANG, Ang, APAF-1, APE, APJ, APP, APRIL, AR, ARC, ART, Qing Artesunate (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-2BMP-2a, BMP-3 osteogenic factor, BMP-4BMP-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), cancer associated antigen, cathepsin A, cathepsin B, cathepsin C / DPPI, cathepsin D, cathepsin E, cathepsin H, cathepsin L, cathepsin O, cathepsin S, cathepsin V, cathepsin X / Z / P, CBL, CCI, CCK2, CCL, CCL1, CCL11, CCL12, CCL13, CCL 14, CCL15, CCL16, CCL17. CCL18, CCL19, CCL2, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9 / 10, CCR, CCR1, CCR10, CCR10, CC R2, 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, C D23, 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, Clostridium botulinum toxin, Clostridium perfringens toxin, CKb8-1, CLC, CMV, CMV UL, CNTF, CNTN-1, COX, C-Ret, CRG-2, CT-1, CTACK, CTGF, CTLA-4, CX3CL1, CX3CR1, CXCL, CXCLl,, CXCL2, CXCL3, CXC L4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCR, CXCR1, CX CR2, CXCR3, CXCR4, CXCR5, CXCR6, cytokeratin tumor-associated antigen, DAN, DCC, DcR3, DC-SIGN, decay-accelerating factor, des(l-3)-IGF-I (brain IGF-1), Dhh, digoxin, DNAM-1, Dnase, Dpp, DPPIV / CD26, Dtk, ECAD, EDA, EDA-A1, EDA-A2, EDAR, EGF, EGFR (ErbB-1), EMA, EMMPRIN, 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, fibrinogen, 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-α1, GFR-α2, GFR-α3, 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), HepB 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, HIVIIIB gp 120V3 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-18 R, IL-23, interferon (INF)-α, INF-β, INF-γ, inhibin, iNOS, insulin A chain, insulin B chain, insulin-like growth factor 1, integrin α2, integrin α3, integrin α4, integrin α4 / βl, integrin, α4 / β7, integrin α5 (αV), integrin α5 / βl, integrin α5 / β3, integrin α6, integrin βl, integrin β2, interferon γ, IP-10, 1-TAC, JE, kallikrein 2, kallikrein 5, kallikrein 6, kallikrein 11, kallikrein 12, kallikrein 14, kallikrein 15, kallikrein LI, kallikrein L2, kallikrein Kallikrein L3, Kallikrein L4, KC, KDR, Keratinocyte Growth Factor (KGF), Laminin 5, LAMP, LAP, LAP (TGF-1), Potential TGF-1, Potential TGF-1bpl, LBP, LDGF, LECT2, Lefty, Lewis-Y Antigen, Lewis-Y Related Antigen, LFA-1, LFA-3, Lfo, LIF, LIGHT, Lipoprotein, LIX, LKN, Lptn, L-Selectin, LT-a, LT-b, LTB4, LTBP-1, Pulmonary Surfactant, Luteinizing Hormone, Lymphotoxin B Receptor, Mac-1, MAdCAM, MAG, MAP2, MARC, MCAM, MCAM, MCK-2, MCP, M-CSF, MDC, Mer, α-metalloproteinase, MGDF receptor, MGMT, MHC (HLA-DR), MIF, MIG, MIP, MIP-1-α, 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, Mullerian-inhibiting substance (MUC18),substance), Mug, MuSK, NAIP, NAP, NCAD, N-cadherin, NCA 90, NCAM, NCAM, Neprilysin, neurotrophin-3, -4 or -6, neurotrophin (Neurturin), neuron growth factor (NGF), NGFR, NGF-β, nNOS, NO, NOS, Npn, NRG-3, NT, NTN, OB, OGG1, OPG, OPN, OSM, OX40L, OX40R, pl50, p95, PADPr, parathyroid hormone, PARC, PARP, PBR, PBSF, PCAD, P-cadherin, PCNA, PDGF , PDGF, PDK-1, PECAM, PEM, PF4, PGE, PGF, PGI2, PGJ2, PIN, PLA2, placental alkaline phosphatase (PLAP), P1GF, PLP, PP14, proinsulin, prorelaxin, protein C, PS, PSA, PSCA, prostate-specific membrane antigen (PSMA), PTEN, PTHrp, Ptk, PTN, R51, RANK, RANKL, RANTES, 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 receptors (e.g., T cell receptor α / β), TdT, TECK, TEM1, TEM5, TEM7, TEM8, TERT, testis P LAP-like alkaline phosphatase, TfR, TGF, TGF-α, TGF-β, TGF-βPan specific, TGF-βRI (ALK-5), TGF-βRII, TGF-βRllb, TGF-βRIII, TGF-βl, TGF-β2, TGF-β3, TGF-β4, TGF-β5, thrombin, thymus Ck-1, thyroid stimulating hormone, Tie, TIMP, TIQ, tissue factor, TMEFF2, Tmpo, TMPRSS2, TNF, TNF-α, TNF-αβ, TNF-β2, TNFc, TNF-RI, TNF-RII, TNFRSF10A (TRAIL Rl Apo-2, DR4), TNFRSFIOB (TRAIL R2DR5, KILLER, TRICK-2A, TRICK-B), TNFRSF10C (TRAILR3DcRl、LIT、TRID)、TNFRSF10D(TRAIL R4DcR2、TRUNDD)、TNFRSF11A(RANK ODF R、TRANCE R)、TNFRSFllB(OPG OCIF、TR1)、TNFRSF12(TWEAK RFN14)、TNFRSF13B(TACI)、TNFRSF13C(BAFF R)、TNFRSF14(WHEM ATAR、HveA、LIGHT R、TR2)、TNFRSF16(NGFR p75NTR)、TNFRSF17(BCMA) TNFRSF18(GITR). AITR)、TNFRSF19(TROY CROWN、TRADE)、TNFRSF19L(RELT)、TNFRSFIA(TNF RI CD120a、p55-60)、TNFRSFIB(TNF RIICD120b, p75-80, TNFRSF26(TNFRH3), TNFRSF3(LTbR TNF RIII, TNFC R), TNFRSF4(OX40 ACT35, TXGP1R), TNFRSF5(CD40p50), TNFRSF6(Fas). Apo-1, APT1, CD95, TNFRSF6B(DcR3M68, TR6), TNFRSF7(CD27), TNFRSF8(CD30), TNFRSF9(4-lBB CD137, ILA), TNFRSF21(DR6), TNFRSF22(DcTRAIL). R2TNFRH2、TNFRST23(DcTRAIL Rl TNFRH1)、TNFRSF25(DR3Apo-3、LARD、TR-3、TRAMP、WSL-1)、TNFSF10(TRAIL Apo-2 division, TL2, TNFSF11 (TRANCE / RANK division ODF, OPG division), TNFSF12 (TWEAK Apo-3 division, DR3 division), TNFSF13 (APRIL TALL2), TNFSF13B (BAFF). LIGHT、TALL1、THANK、TNFSF20)、TNFSF14(LIGHT HVEM fragment, LTg, TNFSF15 (TL1A / VEGI), TNFSF18 (GITR fragment, AITR fragment, TL6), TNFSFIA (TNF-dependent enzyme (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-lBB ligand CD137 ligand), TP-1, t-PA, Tpo, TRAIL, TRAIL R, TRAIL-R1, TRAIL-R2, TRANCE, transfer receptor, TRF, Trk, TROP-2, TSG, TSLP, tumor-associated antigen CA 125, tumor-associated antigen expressing Lewis Y-related carbohydrate carbohydrate), 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 antigens, VLA, VLA-1, VLA-4, VNR integrin, anti-von Willebrand factor (von Willebrands 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 3), hormone receptors and growth factors.

[0113] Multispecific antibodies comprising the anti-CD3 antibodies and antigen-binding fragments disclosed herein can be prepared according to a variety of techniques, including but not limited to recombinant co-expression of two immunoglobulin heavy chain-light chain pairs with different specificities (see, Milstein and Cuello, Nature 305:537 (1983)), WO 93 / 08829, and Traunecker et al., EMBO J. 10:3655 (1991)), "knob-in-hole" engineering (see, e.g., U.S. Pat. No. 5,731,168); immunoglobulin exchanger (also known as Fab domain exchange or cross-Mab format) technology (see, e.g., WO 2009 / 080253; Schaefer et al., Proc. Natl. Acad. Sci. USA, 108:11187-11192 (2011)); engineered electrostatic steering effects for preparing antibody Fc-heterodimer molecules (WO 2009 / 089004A1); cross-linking of two or more antibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980, and Brennan et al., Science, 229:81 (1985)); leucine zippers (see, e.g., Kostelny et al., J. Immunol, 148(5):1547-1553 (1992)); "diabody" technology (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); single-chain Fv (sFv) dimers (see, e.g., Gruber et al., J. Immunol, 152:5368 (1994)); and trispecific antibodies, as described, e.g., in Tutt et al., J. Immunol 147:60 (1991).

[0114] The present disclosure also encompasses modifications of the anti-CD3 antibodies disclosed herein, such modifications comprising one or more amino acid substitutions, insertions and / or deletions in the FR regions and / or CDR regions of the heavy and light chain variable domains. Once obtained, such derivative antibodies and / or antigen-binding fragments can be tested for one or more desired properties, such as improved binding specificity, increased binding affinity, improved developability, etc.

[0115] In some embodiments, the anti-CD3 antibody and / or antigen-binding fragment thereof comprises a heavy chain (HC) sequence, a light chain (LC) sequence, a CDRH3 sequence, a CDRH2 sequence, a CDRH1 sequence, a CDRL3 sequence, a CDRL2 sequence, a CDRL1 sequence and / or a framework sequence. In some embodiments, the anti-CD3 antibody and / or antigen-binding fragment thereof has an amino acid sequence identity of at least about 100%, at least about 99%, at least about 98%, at least about 97%, at least about 96%, at least about 95%, at least about 94%, at least about 93%, at least about 92%, at least about 91%, at least about 90%, at least about 89%, at least about 88%, at least about 87%, at least about 86%, at least about 85%, at least about 84%, at least about 83%, at least about 82%, at least about 80%; and / or all percentages of identity therebetween. In some embodiments, percent identity is measured by any well-known algorithm of sequence identity, such as FASTA, BLAST, or GAP.

[0116] In some embodiments, the residue positions that are not identical differ due to conservative amino acid substitutions. "Conservative amino acid substitutions" are amino acid substitutions in which an amino acid residue is substituted by another amino acid residue containing a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, conservative amino acid substitutions will not substantially change the functional properties of a protein. In the case where two or more amino acid sequences differ from each other due to conservative substitutions, the percentage or degree of similarity can be adjusted upward to correct the conservative nature of the substitution. The means for performing such regulation are well known to those skilled in the art. (See, e.g., Pearson (1994) Methods Mol. Biol. 24: 307-331). Examples of amino acid groups containing side chains with similar chemical properties include 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic-hydroxy side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains: lysine, arginine, and histidine; 6) acidic side chains: aspartic acid and glutamic acid, and 7) sulfur-containing side chains: cysteine ​​and methionine. In some embodiments, conservative amino acid substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine. Alternatively, in some embodiments, conservative substitutions include any changes with positive values ​​in the PAM250 log-likelihood matrix disclosed in the following literature: Gonnet et al. (1992) Science 256:1443-45. In some embodiments, a "moderately conservative" substitution includes any change that has a non-negative value in the PAM250 log-likelihood matrix.

[0117] The substitution of one or more CDR residues or the omission of one or more CDRs is also possible. Antibodies in which one or two CDRs can be assigned to change the binding in the scientific literature have been described. Padlan et al. (1995 FASEB J. 9: 133-139) analyzed the contact area between the antibody and its antigen based on the disclosed crystal structure, and concluded that only about one-fifth to one-third of the CDR residues actually contacted its associated antigen. Padlan also found that many antibodies in which one or two CDRs had zero amino acids in contact with the antigen (see also Vajdos et al. 2002 J Mol Biol 320: 415-428). CDR residues that do not contact the antigen can be identified by molecular modeling and / or empirically from the region of the Kabat CDR outside the Chothia CDR based on previous studies (e.g., residues H60-H65 in CDRH2 are usually not required). If a CDR or its residue is omitted, it is usually replaced by an amino acid occupying a corresponding position in another human antibody sequence or a consensus sequence of such a sequence. The positions within the CDRs for substitution and the amino acids to be substituted can also be selected empirically.

[0118] In certain embodiments, substitutions, insertions or deletions may occur within one or more CDRs of the engineered pH-dependent CD3 binding antibodies described herein, as long as such changes retain pH sensitivity and do not substantially reduce the ability of the antibody to bind its antigen. For example, conservative changes (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity may be made in the CDRs. Such changes may, for example, be located outside of the antigen contact residues in the CDRs. In certain embodiments of the variant VH and VL sequences provided above, each CDR is unchanged, or contains no more than one, two or three amino acid substitutions.

[0119] An effective method for identifying antibody residues or regions that can be targeted for mutagenesis is called "alanine scanning mutagenesis", as described in Cunningham and Wells (1989) Science, 244: 1081-1085. In this method, a residue or a group of target residues (e.g., charged residues such as arg, asp, his, lys and glu) is identified and replaced with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the interaction of the antibody with the antigen is affected. Additional substitutions can be introduced at the amino acid position to demonstrate functional sensitivity to the initial substitution. Alternatively or in addition, the crystal structure of the antigen-antibody complex to identify the contact points between the antibody and the antigen. Such contact residues and adjacent residues can be targeted or eliminated as replacement candidates. Variants can be screened to determine whether they contain desired properties.

[0120] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing one hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include antibodies with an N-terminal methionyl residue. Other insertion variants of antibody molecules include fusions to the N-terminus or C-terminus of the antibody to increase the serum half-life of the antibody (e.g., for ADEPT) or polypeptides.

[0121] As described throughout, the anti-CD3 antibodies and / or antigen-binding fragments thereof as provided herein have favorable developability properties and are therefore relatively developable.

[0122] The term "developable" refers to the extent to which one or more of the plurality of polypeptides has desired characteristics, such as, for example, desired expression in mammalian cells; solubility; viscosity; aggregation; chemical and / or physical stability; desired shelf life; melting temperature; pharmacokinetic profile; circulation half-life; and clearance characteristics. Such characteristics may serve as markers independently; as a combination of a subset of such markers; or as a whole, it is possible that such one or more polypeptides may be successfully developed as therapeutic candidates and ultimately become approved drugs. Therefore, as understood in the art, generally, polypeptides having desired developability characteristics possess, for example, relatively high solubility, relatively low viscosity, relatively low aggregation tendency, relatively high chemical stability, relatively high physical stability, relatively long shelf life, relatively high melting temperature, relatively long circulation half-life, relatively long clearance time, etc. Polypeptides with undesirable developability characteristics possess, for example, relatively low solubility, relatively high viscosity, relatively high aggregation tendency, relatively poor chemical stability, relatively poor physical stability, relatively short shelf life, relatively low melting temperature, relatively short circulation half-life, relatively short clearance time, etc.

[0123] Methods and assays that can be used to determine the extent to which a polypeptide (such as an anti-CD3 antibody and / or antigen-binding fragment thereof described herein) possesses desired developability characteristics are available in the art and include, for example: PSR assays (WO2014 / 179363 and Xu et al., Protein Eng Des Sel, Vol. 26, pp. 663-670 (2013)); SMP and SCP assays, etc.; 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 methods; BVP ELISA assay; AC-SINS assay (Liu et al.; MAbs, Vol. 6, pp. 483-492 (2014)); differential scanning calorimetry; etc. (see, e.g., He et al., J. Pharm. Sci., Vol. 100(4), pp. 1330-1340 (2011); Wagner et al., Pharm. Develop. & Technol (published online 2012; hypertext transfer protocol: informahealthcare.com / doi / abs / 10.3109 / 10837450.2011.649851); Hotz el et al., MAbs, Vol. 4(6), pp. 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)).

[0124] In some embodiments, antibodies identified as possessing reduced developability are detected as such by their interaction with a multispecific reagent ("PSR"), and are therefore referred to as "multispecific" polypeptides. Such multispecific antibodies may be referred to as relatively "non-developable" or relatively "non-developable".

[0125] "Developability profile" refers to an index that can be assigned to an antibody when evaluating the developability of an antibody. A developability profile is a measure or metric that can evaluate, compare and / or rank the developability of anti-CD3 antibodies. Such developability profiles are used as a measure of the degree of interaction between CD3 binders and antibodies comprising them. The degree of interaction can be assessed by any number of means available in the art, which provide output values ​​related to the strength or affinity of the polypeptide to the part to which it is bound. Exemplary means include flow cytometry means, such as FACS; ELISA; quantitative immunoaffinity assays or immunoprecipitation assays; mammalian two-hybrid or yeast two-hybrid assays, etc. In the context of FACS, as shown in the examples, the degree of interaction between the polypeptides and PSRs in the multiple polypeptides can be determined by generating a mean fluorescence intensity (MFI) for each polypeptide-PSR interaction detected, and then sorting the MFI in ascending or descending order, thereby ranking the polypeptides in the multiple polypeptides according to the relative degree of interaction between each detected polypeptide and PSR. Such a ranking provides a ranking of the polypeptides of the plurality of polypeptides, allowing for easy identification of those polypeptides that possess enhanced developability, as well as those polypeptides that possess decreased developability.

[0126] The developability profile can also take the form of a normalized score, for example by normalizing the developability of an anti-CD3 antibody described herein to the developability of a standard (or control) antibody (eg, an anti-HEL antibody).

[0127] In certain embodiments, the engineered pH-dependent CD3 binding domains of the present invention and antibodies comprising them can be further modified to contain additional non-proteinaceous moieties known in the art and readily available. Suitable moieties for antibody derivatization include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), copolymers of ethylene glycol / propylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers), and dextran or poly (n-vinyl pyrrolidone) polyethylene glycol, polyethylene glycol homopolymers, polypropylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde has advantages in manufacturing due to its stability in water. The polymer can have any molecular weight and can be branched or unbranched. The number of polymers attached to the antibody can vary, and if more than one polymer is attached, they can be the same or different molecules. In general, the number and / or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the specific properties or functions of the antibody to be improved, whether the antibody derivative is used for therapy under defined conditions, etc.

[0128] In certain embodiments, the engineered pH-dependent CD3 binding domains and antibodies comprising the same exhibit an enhanced developability profile. The developability profile of the anti-CD3 antibody is obtained by performing one or more of a PSR assay; an SCP assay; an AC-SINS assay; an ELISA; a DSF assay; a Tm assay; a HIC assay; a CIC assay; or a combination thereof.

[0129] In other embodiments, the anti-CD3 antibodies and / or antigen-binding fragments thereof as described herein exhibit a multispecific agent (PSR) score between about 0.0 and about 0.45. In some embodiments, the PSR is between about 0.0 and about 0.4. In some embodiments, the PSR is between about 0.0 and about 0.35. In some embodiments, the PSR is between about 0.0 and about 0.3. In some embodiments, the PSR is between about 0.0 and about 0.25. In some embodiments, the PSR is between about 0.0 and about 0.2. In some embodiments, the PSR is between about 0.0 and about 0.15. In some embodiments, the PSR is between about 0.0 and about 0.1. In some embodiments, a score of 0.0-0.1 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 0.66-1.00 is a "high PSR". In some embodiments, a high PSR score indicates reduced (or poor) developability. Generally, the lower the PSR score, the more favorable the developability of the antibody.

[0130] In still other embodiments, an anti-CD3 antibody or antigen-binding fragment thereof as described herein exhibits a HIC score of less than about 10.5 minutes (clean to low HIC score). In some embodiments, the HIC score is between about 10.5 minutes and 11.5 minutes (medium HIC score). In some embodiments, the HIC score is greater than about 11.5 minutes (high HIC score). Generally, the lower the HIC score, the more favorable the developability of the antibody.

[0131] In yet other embodiments, the anti-CD3 antibodies and / or antigen-binding fragments thereof as described herein exhibit a SEC score of less than about 95%, indicating that the antibody is monomeric, ie, does not aggregate.

[0132] In still other embodiments, the anti-CD3 antibodies and / or antigen-binding fragments thereof as described herein exhibit a Tm of less than about 65°C.

[0133] In some embodiments, the anti-CD3 antibodies and / or antigen-binding fragments thereof as described herein can be further modified to minimize effector functions, such as silencing the Fc.

[0134] "Effector function" refers to the biological activity attributable to the Fc region of an antibody, which varies with the antibody isotype. 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.

[0135] "Fc region" is the C-terminal region of the immunoglobulin heavy chain containing at least a portion of the constant region, including native sequence Fc regions and variant Fc regions. The human IgG heavy chain Fc region may extend from Cys226 or from 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 is according to the EU numbering system, also referred to as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition Public Health Service, National Institutes of Health, Bethesda, Md., 1991.

[0136] 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, thereby generating Fc region variants (see, e.g., US2012 / 0251531). The Fc region variant may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) comprising an amino acid modification (e.g., substitution) at one or more amino acid positions.

[0137] In certain embodiments, the present disclosure contemplates anti-CD3 antibody variants that possess some but not all effector functions, making them desirable candidates for applications where in vivo antibody half-life is important but certain effector functions (such as complement and ADCC) are unnecessary or deleterious. In vitro and / or in vivo cytotoxicity assays can be performed to confirm reduction / abatement of CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to ensure that the antibody lacks FcγR binding (and therefore may lack ADCC activity) but retains FcRn binding ability. Primary cells used to mediate ADCC (e.g., NK cells) express only FcγRIII, whereas monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays for evaluating ADCC activity of molecules of interest are described in U.S. Pat. No. 5,500,362 (see, e.g., Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); U.S. Pat. No. 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assays can be used (see, e.g., ACTI for flow cytometry). TM Nonradioactive cytotoxicity assay (Cell Technology, Inc. Mountain View, Calif.); and CytoTox Non-radioactive cytotoxicity assay (Promega, Madison, Wis.). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively or in addition, ADCC activity of the molecule of interest can be assessed in vivo, for example, in an animal model such as that disclosed in Clynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). A C1q binding assay can also be performed to confirm that the antibody cannot bind to C1q and therefore lacks CDC activity. See, for example, C1q and C3c binding ELISAs in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, a CDC assay can be performed (see, e.g., Gazzano-Santoro et al. J. Immunol Methods 202: 163 (1996); Cragg, MS et al. Blood. 101: 1045-1052 (2003); and Cragg, MS and MJ Glennie Blood. 103: 2738-2743 (2004)). FcRn binding and in vivo clearance / half-life determinations can also be performed using methods known in the art (see, e.g., Petkova, SB et al. Int'l. Immunol 18(12): 1759-1769 (2006)).

[0138] In some embodiments, antibodies with reduced effector function include antibodies with substitutions of one or more of Fc region residues 238, 265, 269, 270, 297, 327, and 329 (U.S. Pat. Nos. 6,737,056 and 8,219,149). In some embodiments, Fc mutants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc mutants with substitutions of residues 265 and 297 to alanine (U.S. Pat. Nos. 7,332,581 and 8,219,149).

[0139] In other embodiments, the anti-CD3 antibodies and / or antigen-binding fragments thereof as described herein are further modified to include a masking agent, such as a polypeptide mask, attached via a cleavable linker.

[0140] In some embodiments, an anti-CD3 antibody and / or antigen-binding fragment thereof as described herein is conjugated to a therapeutic moiety to form an immunoconjugate. An "immunoconjugate" is an antibody conjugated to one or more heterologous molecules (e.g., an antibiotic, a second anti-CD3 antibody, a vaccine or toxoid, or any other therapeutic moiety).

[0141] In certain embodiments, the anti-CD3 antibodies and / or antigen-binding fragments thereof described herein are altered to increase or decrease the extent to which the antibody is glycosylated. Glycosylation sites can be conveniently added or deleted to the anti-CD3 antibodies of the present disclosure by altering the amino acid sequence so as to create or remove one or more glycosylation sites.

[0142] Production of anti-CD3 antibodies and their antigen-binding fragments

[0143] Anti-CD3 antibodies and / or antigen-binding fragments thereof can be produced using recombinant methods. For example, isolated nucleic acids encoding anti-CD3 antibodies as described herein are provided. Such nucleic acids can encode an amino acid sequence comprising the VL of the antibody and / or an amino acid sequence comprising the VH (e.g., the light chain and / or heavy chain of the antibody). In another embodiment, one or more vectors (e.g., expression vectors) comprising such nucleic acids are provided. In another embodiment, a host cell comprising such nucleic acids is provided. In one such embodiment, the host cell comprises (e.g., has been transformed with the following items): (1) a vector comprising a nucleic acid sequence encoding an amino acid sequence comprising the VL of the antibody and an amino acid sequence comprising the VH of the antibody, or (2) a first vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody, and a second vector comprising a nucleic acid encoding an amino acid sequence comprising the VH of the antibody. In one embodiment, the host cell is eukaryotic, such as a Chinese hamster ovary (CHO) cell or a lymphocyte (e.g., a Y0, NS0, Sp20 cell). In one embodiment, a method of preparing an anti-CD3 antibody is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the antibody as provided above under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium).

[0144] The term "host cell" refers to a cell into which an exogenous nucleic acid sequence has been introduced, including the progeny of such cells. Host cells include transformants and transformed cells, including the primary transformed cell and progeny derived therefrom, regardless of the number of passages.

[0145] For recombinant production of anti-CD3 antibodies, nucleic acids encoding antibodies, such as those described above, are isolated and inserted into one or more vectors for further cloning and / or expression in host cells. Such nucleic acids can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that can specifically bind to genes encoding antibody heavy and light chains).

[0146] Suitable host cells for cloning and / or expressing vectors encoding antibodies include prokaryotes or eukaryotic cells. For example, particularly when glycosylation and Fc effector functions are not required, antibodies can be produced in bacteria. Regarding the expression of antibody fragments and polypeptides in bacteria, see, for example, U.S. Patent Nos. 5,648,237, 5,789,199 and 5,840,523. (See also Charlton, Methods in Molecular Biology, Vol. 248 (BKCLo ed., Humana Press, Totowa, NJ, 2003), pp. 245-254, describing the expression of antibody fragments in Escherichia coli.) After expression, antibodies can be separated from bacterial cell paste in soluble fractions and can be further purified. In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are suitable cloning or expression hosts for vectors encoding antibodies, including fungi and yeast strains whose glycosylation pathways have been "humanized", so as to produce antibodies with partial or complete human glycosylation patterns. See, e.g., Gerngross, Nat. Biotech. 22: 1409-1414 (2004), and Li et al., Nat. Biotech. 24: 210-215 (2006); WO 2009 / 036379; WO 2010 / 105256; and WO 2012 / 009568.

[0147] Plant cell cultures can also be used as hosts. See, e.g., U.S. Pat. Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing PLANTIBODIES for producing antibodies in transgenic plants). TMTechniques). Viral cells can also be used as hosts. For example, mammalian cell lines adapted to growth in suspension may be useful. Other examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney line (such as 293 or 293 cells described in, e.g., Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK); mouse Sertoli cells (such as TM4 cells described in, e.g., Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK); buffalo rat liver cells (BRL 3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary tumor (MMT 060562); TRI cells, such as described in, e.g., Mather et al., Annals of NYAcad.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)); and myeloma cell lines such as Y0, NS0 and Sp2 / 0. For a review of certain mammalian host cell lines suitable for antibody production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKCLo ed., Humana Press, Totowa, NJ), pp. 255-268 (2003).

[0148] Anti-CD3 antibodies and / or antigen-binding fragments thereof can be identified, screened, selected or characterized by various assays known in the art (e.g., ELISA, Western blot, etc.) for their physical / chemical properties and / or biological activity, or competitive assays can be used to identify antibodies that compete with the anti-CD3 antibodies of the present disclosure for binding to CD3. In an exemplary competitive assay, fixed CD3 is incubated in a solution containing a first labeled antibody that binds to CD3 and a second unlabeled antibody that is being 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, fixed CD3 is incubated in a solution containing a first labeled antibody instead of a second unlabeled antibody. After incubation under conditions that allow the first antibody to bind to CD3, excess unbound antibodies are removed and the amount of label associated with fixed CD3 is measured. If the amount of label associated with fixed CD3 in the test sample is substantially reduced relative to the control sample, it indicates that the second antibody competes with the first antibody for binding to CD3. See, e.g., Harlow and Lane (1988) Antibodies: A Laboratory Manual. Chapter 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

[0149] Anti-CD3 antibodies and / or antigen-binding fragments thereof having biological activity can be identified using standard methods. Biological activity can include, for example, in vivo, in vitro, or ex vivo binding to CD3 on the surface of a T cell. In the case of a multispecific anti-CD3 antibody (such as a bispecific antibody having one arm that binds to CD3 and another arm that binds to a different target (e.g., a cell surface antigen, such as a tumor antigen), biological activity can also include effector cell activation (such as CD8+ and / or CD4+ T cell activation), effector cell population expansion (i.e., an increase in T cell count), target cell population reduction (i.e., a reduction in the cell population expressing the second biomolecule on its cell surface), and / or target cell killing.

[0150] Diagnostic and therapeutic uses of anti-CD3 antibodies and antigen-binding fragments thereof

[0151] The anti-CD3 antibodies and / or antigen-binding fragments described herein can be used for diagnosis and / or detection. As used herein, "detection" encompasses quantitative or qualitative detection.

[0152] In certain embodiments, labeled anti-CD3 antibodies are provided. Anti-CD3 antibodies and / or antigen-binding fragments thereof as described herein may include a label or moiety that is detected directly (such as fluorescent, chromogenic, electron-focused, chemiluminescent, and radioactive labels) or indirectly (such as enzymes or ligands). Non-limiting exemplary labels include radioisotopes such as 32P, 14C, 125I, 3H, and 131I; fluorophores such as rare earth chelates or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luciferase (e.g., firefly luciferase and bacterial luciferase (U.S. Pat. No. 4,737,456)), luciferin, 2,3-dihydrophthalazinedione, horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, sugar oxidases (e.g., glucose oxidase, galactose oxidase), and glucose-6-phosphate dehydrogenase; heterocyclic oxidases (such as uricase and xanthine oxidase) coupled to enzymes that employ hydrogen peroxide to oxidize dye precursors such as HRP, lactoperoxidase, or microperoxidase; biotin / avidin; rotation tags; bacteriophage tags; stable free radicals; and the like.

[0153] The CD3 antibodies and / or antigen-binding fragments thereof as described herein and pharmaceutical compositions of such antibodies can be used in therapeutic methods. In one embodiment, the anti-CD3 antibodies and / or antigen-binding fragments thereof as described herein or pharmaceutical compositions comprising such antibodies can be used to treat or delay the progression of cell proliferative disorders or autoimmune disorders. In some embodiments, anti-CD3 antibodies and antigen-binding fragments thereof can be used to treat cancer. Tumor cells typically have an extracellular pH of about 6.3-6.5; the anti-CD3 antibodies and antigen-binding fragments described herein promote preferential CD3 binding at low (lower) pH values ​​(e.g., around pH 6), and thereby promote binding and activity in and around the tumor microenvironment. In some embodiments, the use of anti-CD antibodies and antigen-binding fragments thereof can produce selective and sustained cytotoxic activity at or around the tumor site, thereby reducing or eliminating off-target effects.

[0154] "Disorder" refers to any condition or disease that would benefit from treatment, including, but not limited to, chronic and acute disorders or diseases, including those pathological conditions that predispose the mammal to the disorder in question.

[0155] The terms "cell proliferative disorder" and "proliferative disorder" refer to disorders that are associated with some degree of abnormal cell proliferation. Cell proliferative disorders include cancer, such as tumors.

[0156] As used herein, "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all precancerous and cancerous cells and tissues.

[0157] "Cancer" refers to the physiological condition in mammals characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies; more specific examples include squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), lung cancer (including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma), peritoneal cancer, hepatocellular carcinoma, stomach or gastric cancer (including gastrointestinal cancer and gastrointestinal stromal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, urinary tract cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney or renal cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, penis cancer, melanoma, superficial spreading melanoma, lentigo malignant melanoma, acral lentigo melanoma, nodular melanoma, multiple myeloma The disease is characterized by the presence of a variety of neoplasms, including low-grade / follicular non-Hodgkin lymphoma (NHL); small lymphocytic (SL) NHL; intermediate-grade / follicular NHL; intermediate-grade diffuse NHL; high-grade immunoblastic NHL; high-grade lymphoblastic NHL; high-grade small non-lytic cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's macroglobulinemia); chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); hairy cell leukemia; chronic myeloblastic leukemia; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with nevus hamartomatosis, edema (such as that associated with brain tumors), Meigs syndrome, brain and head and neck cancers, and related metastases. In certain embodiments, cancers suitable for treatment by the antibodies of the present disclosure include breast cancer, colorectal cancer, rectal cancer, non-small cell lung cancer, glioblastoma, non-Hodgkin's lymphoma (NHL), renal cell carcinoma, prostate cancer, liver cancer, pancreatic cancer, soft tissue sarcoma, Kaposi's sarcoma, carcinoid carcinoma, head and neck cancer, ovarian cancer, mesothelioma and multiple myeloma. In some embodiments, the cancer is selected from: small cell lung cancer, glioblastoma, neuroblastoma, melanoma, breast cancer, gastric cancer, colorectal cancer (CRC) and hepatocellular carcinoma. 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 is selected from a class of mature B cell cancers that excludes Hodgkin lymphoma but includes germline B-cell-like (GCB) DLBCL, activated B-cell-like (ABC) DLBCL, follicular lymphoma (FL), mantle cell lymphoma (MCL), acute myeloid leukemia (AML), chronic lymphoid leukemia (CLL), marginal zone lymphoma (MZL), small lymphocytic leukemia (SLL), lymphoplasmacytic lymphoma (LL), Waldenstrom's macroglobulinemia (WM), central nervous system lymphoma (CNSL), Burkitt's 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 variants, Waldenstrom's macroglobulinemia, heavy chain disease, alpha heavy chain disease, gamma heavy chain disease, mu heavy chain disease, plasma cell myeloma, solitary plasmacytoma of bone, extraosseous plasmacytoma, mucosa-associated lymphoma Extranodal marginal zone lymphoma of barbed tissue (MALT lymphoma), nodal marginal zone lymphoma, nodal marginal zone lymphoma of children, follicular lymphoma of children, primary cutaneous follicle center lymphoma, T cell / histiocyte-rich large B cell lymphoma, primary DLBCL of the CNS, primary cutaneous DLBCL (leg type), EBV-positive DLBCL of the elderly, DLBCL associated with chronic inflammation, lymphomatoid granuloma, 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 lymphadenopathy (Castleman) disease, primary effusion lymphoma; unclassifiable B-cell lymphoma with features intermediate between diffuse large B-cell lymphoma and Burkitt's lymphoma, and unclassifiable B-cell lymphoma with features intermediate between diffuse large B-cell lymphoma and classical Hodgkin's lymphoma.

[0158] As used herein, "treatment" or "treat" or "treating" refers to a clinical intervention that attempts to alter the natural course of the individual being treated, and can be used for prevention or performed during clinical pathology. The desired effects of treatment include, but are not limited to, prevention of the occurrence or recurrence of the disease, alleviation of symptoms, reduction of any direct or indirect pathological consequences of the disease, prevention of metastasis, reduction of the rate of disease progression, improvement or alleviation of the disease state, and relief or improved prognosis.

[0159] As used herein, the terms "prevent," "preventing," and "prevention" refer to preventing or inhibiting the development or onset of a disorder or disease.

[0160] As used herein, the terms "ameliorate" and "alleviate" refer to a decrease in the severity or reduction in a disorder or any symptom thereof.

[0161] In some embodiments, the antibodies disclosed herein are used to delay the development of a disorder or disease or to delay the progression of a disorder or disease. As used herein, "delayed progression" of a disorder or disease means to delay, hinder, slow down, prevent, stabilize and / or postpone the development of a disease or disorder (e.g., a cell proliferative disorder, such as cancer). The delay can have different lengths of time, depending on the disease being treated and / or the history of the individual.

[0162] An effective amount of such an antibody or composition can be administered to an individual suffering from cancer or arthritis, rheumatoid arthritis, colitis, inflammatory bowel disease, autoimmune type I diabetes, etc. An "effective amount" of an anti-CD3 antibody disclosed herein or a composition (e.g., a pharmaceutical composition) comprising such an antibody is at least the minimum amount required to achieve a desired therapeutic or preventive result, such as a measurable improvement or prevention of a particular disorder (e.g., a cell proliferative disorder, such as cancer), preferably with minimal or no toxic or deleterious effects. The effective amount can vary depending on, among other things, the disease state, age, sex, and weight of the patient, and the ability of the antibody (or antigen-binding fragment thereof) to elicit a desired response in the individual and, in some cases, due to co-administration of one or more additional therapeutic agents.

[0163] In some embodiments, anti-CD3 antibodies and / or antigen-binding fragments thereof as described herein can be used to enhance the immune function of individuals suffering from cell proliferative disorders or autoimmune disorders. After administration, such antibodies or compositions can enhance the immune function of individuals suffering from cell proliferative disorders or autoimmune disorders by activating effector cells (e.g., T cells, such as CD8+ and / or CD4+T cells, including Treg), amplifying (increasing) effector cell populations, reducing target cell populations (e.g., cells expressing a second biomolecule recognized by an anti-CD3 antibody of the present disclosure (such as a bispecific antibody)), and / or killing target cells (e.g., target tumor cells).

[0164] Anti-CD3 antibodies and / or antigen-binding fragments thereof as disclosed herein can be used to treat disorders including, but not limited to, proliferative disorders, oncological disorders, immuno-oncological disorders, neurological disorders, cognitive disorders, neurodegenerative disorders, autoimmune disorders. In one embodiment, an effective amount of such anti-CD3 antibody, alone or in combination with at least one additional agent, can be administered to an individual suffering from such a disorder. Such an "individual" can be a mammal, and in particular a human.

[0165] Non-limiting exemplary additional therapeutic agents include chemotherapeutic agents, antibody-drug conjugates (ADC) and / or biomodifiers. Chemotherapeutic agents can be selected from cyclophosphamide, doxorubicin, vincristine and prednisolone (CHOP). ADC can be selected from anti-CD79b antibody drug conjugates (such as anti-CD79b-MC-vc-PAB-MMAE or anti-CD79b antibody drug conjugates described in U.S. Patent No. 8,088,378 and / or US2014 / 0030280 or polotuzumab MMAE conjugates (polatuzumab vedotin)), anti-CD19 antibody drug conjugates, anti-CD22 antibody drug conjugates, anti-CD45 antibody drug conjugates and anti-CD32 drug conjugates. Biomodifiers can be selected from BCL-2 inhibitors (such as GDC-0199 / ABT-199); lenalidomide PI3K-δ inhibitors (such as idelalisib) ); PD-1 axis binding antagonist; agonist (e.g., agonist antibody to an activating co-stimulatory molecule, such as CD40, CD226, CD28, OX40 (e.g., AgonOX), GITR, CD137 (also known as TNFRSF9, 4-1BB, or ILA), CD27 (e.g., CDX-1127), HVEM, or CD127); antagonist (e.g., antagonist antibody to an inhibitory co-stimulatory molecule, such as CTLA-4 (also known as CD152), PD-1, TIM-3, BTLA, VISTA, LAG-3, B7-H3, B7-H4, IDO (e.g., 1-methyl-D-tryptophan (also known as 1-D-MT)), TIGIT, MICA / B, GITR (e.g., TRX518) or arginase); ipilimumab (also known as MDX-010, MDX-101, or ); tremelimumab (also known as ticilimumab or CP-675,206); urelumab (also known as BMS-663513); MGA271; antagonists to TGFβ (e.g., metilimumab (also known as CAT-192), fresolimumab (also known as GC1008), LY2157299k); and adoptive transfer of T cells (e.g., cytotoxic T cells or CTLs) expressing a chimeric antigen receptor (CAR) (e.g., adoptive transfer of T cells comprising a dominant negative TGFβ receptor (e.g., a dominant negative TGFβ type II receptor)).

[0166] Anti-CD3 antibodies and / or antigen-binding fragments thereof as disclosed herein can be used to enhance immune function in individuals with a disorder (e.g., humans, individuals with such a disorder). In one embodiment, a method of enhancing immune function comprises administering to an individual an effective amount of an anti-CD3 antibody to activate effector cells (e.g., T cells, such as CD8+ and / or CD4+ T cells), expand (increase) effector cell populations, reduce target cell populations, and / or kill target cells (e.g., target tumor cells).

[0167] In a further aspect, a pharmaceutical formulation comprising an anti-CD3 antibody and / or antigen-binding fragment as described herein is also provided, e.g., for use in any of the above-described methods of treatment. A "pharmaceutical formulation" refers to a preparation that is in such a form as to permit the biological activity of the active ingredient contained therein (such as an anti-CD3 antibody as described herein) to be effective and preferably does not contain additional components that are unacceptably toxic to the subject to which the formulation is to be administered.

[0168] 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 an ingredient 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.

[0169] The antibodies disclosed herein can be used alone or in combination with other agents for therapy, for example, anti-CD3 antibodies and / or antigen-binding fragments thereof can 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 for radiation therapy, an anti-angiogenic agent, an apoptotic agent, an anti-tubulin agent, or other agents such as epidermal growth factor receptor (EGFR) antagonists (e.g., tyrosine kinase inhibitors), HER1 / EGFR inhibitors (e.g., erlotinib (Tarceva TM )), platelet-derived growth factor inhibitors (eg, Gleevec TM (imatinib mesylate)), a COX-2 inhibitor (e.g., celecoxib), an interferon, a cytokine, an antibody other than an anti-CD3 antibody of the present disclosure (such as an antibody that binds one or more of the following targets: ErbB2, ErbB3, ErbB4, PDGFR-β, BIyS, APRIL, BCMA VEGF, or VEGF receptor, TRAIL / Apo2, PD-1, PD-L1, PD-L2), or another biologically active or organic chemical agent.

[0170] In some embodiments, the disclosure provides a method wherein the additional therapeutic agent is a glucocorticoid. In one embodiment, the glucocorticoid is dexamethasone.

[0171] Such combination therapies described above encompass both combined administration (wherein two or more therapeutic agents are included in the same or separate formulations) and separate administration (in which case administration of the disclosed antibody can occur prior to, concurrently with, and / or after administration of the additional therapeutic agent or agents. In one embodiment, administration of the anti-CD3 antibody and administration of the additional therapeutic agent occur within about one month of each other, or within about one, two, three, four, five, or six days. An anti-CD3 antibody of the disclosure (e.g., a bispecific anti-CD3 antibody of the disclosure that binds to CD3 and a second biomolecule (e.g., a cell surface antigen, e.g., a tumor antigen) such as a TDB antibody of the disclosure or a variant thereof) can also be used in combination with radiation therapy.

[0172] The antibodies of the present disclosure (and / or any additional therapeutic agent) can be administered by any suitable means, including parenteral, intrapulmonary and intranasal administration, and (if local treatment is required) intralesional administration. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. In some embodiments, the antibody is administered by subcutaneous administration. In some embodiments, compared with the same anti-CD3 antibody administered by intravenous injection, the anti-CD3 antibody administered by subcutaneous injection shows less toxic reactions in patients. The dosage can be by any suitable route, for example, by injection, such as intravenous or subcutaneous injection, depending in part on whether the administration is short-term or long-term. Various dosing schedules are contemplated herein, including but not limited to single or multiple administrations, push administrations, and pulse infusions at different time points.

[0173] The antibodies disclosed herein will be formulated, dosed and applied in accordance with good medical practice. Factors considered in this context include the specific disorder being treated, the specific mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the delivery site of the medicament, the method of administration, the timetable for administration, and other factors known to the physician. The antibody need not but may optionally be formulated with one or more agents currently used to prevent or treat the disorder in question. The effective amount of such other agents depends on the amount of the antibody present in the formulation, the type of disorder or treatment, and other factors discussed above. These are generally used with the same dosage and route of administration as described herein, or about 1% to 99% of the dosage described herein, or with any dosage and any route determined to be appropriate empirically / clinically.

[0174] For the prevention or treatment of disease, the appropriate dosage of the antibodies of the present disclosure (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease to be treated, the type of antibody, the severity and course of the disease, whether the antibody is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the antibody, and the judgment of the attending physician. The antibody is suitably administered to the patient at one time or over a series of treatments.

[0175] As a general proposition, a therapeutically effective amount of an anti-CD3 antibody administered to a human will be in the range of about 0.01 to about 100 mg / kg of the patient's body weight, whether by one or more administrations. In some embodiments, the antibody used is administered, for example, at 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 per day. In one embodiment, the anti-CD3 antibodies described herein are administered to humans at a dose of about 100 mg, about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, about 1000 mg, about 1100 mg, about 1200 mg, about 1300 mg, or about 1400 mg on day 1 of a 21-day cycle. The dose can be administered as a single dose or as multiple doses (e.g., 2 or 3 doses) such as infusion. For repeated administration over several days or longer, depending on the condition, treatment will generally continue until the desired disease symptom suppression occurs. An exemplary dose of the antibody will be in the range of about 0.05 mg / kg to about 10 mg / kg. Thus, one or more doses of about 0.5 mg / kg, 2.0 mg / kg, 4.0 mg / kg, or 10 mg / kg (or any combination thereof) can be administered to the patient. Such doses may be administered intermittently, e.g., once a week or every three weeks (e.g., so that the patient receives about two to about twenty doses, or, e.g., about six doses of an anti-CD3 antibody). An initial higher loading dose may be administered, followed by one or more lower doses. The progress of such therapy is easily monitored by conventional techniques and assays.

[0176] In some embodiments, the method of the present disclosure may also include additional therapy. Additional therapy may be radiotherapy, surgery, chemotherapy, gene therapy, DNA therapy, virotherapy, RNA therapy, immunotherapy, bone marrow transplantation, nanotherapy, monoclonal antibody therapy, or a combination of the foregoing. Additional therapy may be in the form of adjuvant or neoadjuvant therapy. In some embodiments, additional therapy is the administration of small molecule enzymatic inhibitors or anti-metastatic agents. In some embodiments, additional therapy is the administration of side effect limiters (e.g., agents intended to reduce the occurrence and / or severity of treatment side effects, such as anti-nausea agents, etc.). In some embodiments, additional therapy is radiotherapy. In some embodiments, additional therapy is surgery. In some embodiments, additional therapy is a combination of radiotherapy and surgery. In some embodiments, additional therapy is gamma ray irradiation. In some embodiments, additional therapy may be the separate administration of one or more therapeutic agents described above.

[0177] In another aspect of the present disclosure, an article containing materials that can be used to treat, prevent and / or diagnose the disorders described above is provided. The article includes a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc. The container can be formed of various materials such as glass or plastic. The container contains a composition that can effectively treat, prevent and / or diagnose the condition by itself or in combination with another composition, and can have a sterile access port (for example, the container can be an intravenous infusion bag or a vial with a stopper that can be penetrated by a hypodermic needle). At least one active agent in the composition is an antibody of the present disclosure. The label or package insert indicates that the composition is used to treat a selected condition. In addition, the article may include (a) a first container containing a composition therein, wherein the composition includes an antibody of the present disclosure; and (b) a second container containing a composition therein, wherein the composition includes an additional cytotoxic agent or other therapeutic agent. The article in this embodiment of the present disclosure may also include a package insert indicating that the composition can be used to treat a specific condition. Alternatively or additionally, the article of manufacture may further comprise a second (or third) container comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. It may also contain other materials desired from a commercial and user perspective, including other buffers, diluents, filters, needles, and syringes.

[0178] Therefore, it is also contemplated to manufacture and / or prepare pharmaceutical compositions comprising anti-CD3 antibodies and / or antigen-binding fragments as disclosed herein. The compositions can 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.).

[0179] In some embodiments, a pharmaceutical composition comprising an anti-CD3 antibody and / or antigen-binding fragment thereof as described herein is prepared, for example, by mixing such an antibody having the desired degree of 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, optionally prepared for modified (e.g., sustained) release. Exemplary lyophilized antibody formulations are described in U.S. Pat. No. 6,267,958. Aqueous antibody formulations include those disclosed in U.S. Pat. No. 6,171,586 and WO2006 / 044908, the latter formulation comprising a histidine-acetate buffer.

[0180] Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphates, citrates and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethylammonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl parabens; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polyols. peptides; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates (including glucose, mannose or dextrin); chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein also include interstitial drug dispersants such as soluble neutral active hyaluronidase glycoprotein (sHASEGP), for example, human soluble PH-20 hyaluronidase glycoprotein, such as rHuPH20 ( Baxter International, Inc.). Certain exemplary sHASEGPs, including rHuPH20, and methods of use are described in U.S. Patent Publication Nos. 2005 / 0260186 and 2006 / 0104968.

[0181] Such formulations may contain more than one active ingredient necessary for the particular indication being treated, preferably with complementary activities that do not adversely affect each other and those active ingredients present in amounts effective for the intended purpose. For example, it may be desirable to further provide an additional therapeutic agent (e.g., a chemotherapeutic agent, a cytotoxic agent, a growth inhibitory agent and / or an anti-hormonal agent).

[0182] The active ingredient may be embedded in microcapsules such as hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules in colloidal drug delivery systems such as liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules or in macroemulsions, prepared, for example, by coacervation techniques or by interfacial polymerization. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. ed. (1980).

[0183] Example

[0184] Example 1: Construction of an engineered pH-dependent CD3 library

[0185] The combinatorial histidine replacement library was derived from the parent anti-CD3 antibody clone ADI-26906 (antibody number 1 of Table 1). ADI-26906 was originally disclosed in PCT / US2018 / 031705, which is hereby incorporated by reference in its entirety (ADI-26906 is not pH engineered). Histidine was incorporated using three library designs: 1) H3+L3 skipping double plus L1 single or double histidine (His) replacement (with and without NNK variegation adjacent to His), resulting in a theoretical diversity of 3.4x10 5 ; 2) Pre-made H1 / H2 diversity library plus H3 jump doublets, resulting in a theoretical diversity of 6.8x10 8 , and 3) H3+L3NNK / His or His / NNK walking singletons, resulting in a theoretical diversity of 1.2x 10 5 Libraries were generated and propagated as previously described (see, e.g., 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 CDRs. However, it is contemplated that substitutions can also be designed in FRs. Sequence analysis of variants from each library showed a total of 0-6 His substitutions per variant VH or VK.

[0186] Five rounds of selection were performed using the three libraries against the biotinylated CD3 antigen. For the first round of selection of the H1 / H2 plus H3 libraries, magnetic bead sorting using the Miltenyi MACS system was performed essentially as described (Siegel et al., J Immunol Methods. 2004 Mar; 286(1-2): 141-53). Briefly, approximately 10 9 Yeast cells were incubated with 100nM biotinylated CD3 antigen at pH 6.0 in PBS containing 0.1% BSA at room temperature for 15 minutes. After washing once with 50mL ice-cold washing buffer, the cell pellet was resuspended in 40mL washing buffer, and 500μl streptavidin microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany. Catalog number 130-048-101) were added to the yeast and incubated at 4°C for 15 minutes. Next, the yeast was precipitated, resuspended in 5mL washing buffer, and loaded onto MACS LS columns (Miltenyi Biotec, Bergisch Gladbach, Germany. Catalog number 130-042-401). After loading 5mL, the column was washed three times with 3ml FACS washing buffer. The column was then removed from the magnetic field, and the yeast was eluted with 5mL growth medium and then grown overnight. For the two lower diversity libraries, the first round of selection was performed using flow cytometry (FACS). 9 Yeast cells / library) were incubated with 0.25 mL of 100 nM biotinylated CD3 antigen at pH 6.0 in FACS wash buffer PBS containing 0.1% BSA at pH 6.0 for 15 minutes at room temperature. Yeast were washed with FACS buffer and labeled for sorting.

[0187] After the first round of MACS or FACS, four rounds of sorting were performed using FACS and pH-switch selection methods (see Figure 1).

[0188] Purified CD3 protein antigen was biotinylated using the EZ-Link Sulfo-NHS-Biotinylation Kit (Thermo Scientific). The CD3 antigen was concentrated to approximately 1 mg / mL and the buffer was exchanged into PBS before adding a 1:7.5 molar ratio of the biotinylation reagent (EZ-Link Sulfo-NHS-Biotinylation Kit, Thermo Scientific, catalog number 21425). The mixture was kept at 4°C overnight before another buffer exchange to remove free biotin in solution. Biotinylation was confirmed by binding of the labeled protein to a streptavidin sensor on the ForteBio. Successful biotinylation of the CD3 protein antigen was confirmed by detectable binding to a streptavidin-linked biosensor mounted on a ForteBio OctetTM Red384 interferometer (PallForteBio, Menlo Park, CA) according to the manufacturer's guidelines (data not shown). In CD3 presaturation method #1 (shown in Figure 1A Yeast cells were pre-saturated with native (non-biotinylated) CD3 antigen at pH 7.4 for 10 minutes in PBS (pH 7.4). Next, yeast cells were washed at pH 7.4 and incubated in pH 6.0 medium for 10 minutes to allow antigen dissociation. Control cells were washed and incubated at pH 7.4. Finally, yeast cells were incubated with biotinylated CD3 antigen at pH 6. Figure 1A The control cells were incubated with biotinylated CD3 antigen at pH 7.4. The labeled binders at pH 6 were then sorted and characterized. Figure 1B In the experiment, yeast cells were pre-saturated with native CD3 antigen at pH 6.0 for 10 minutes in 4% paraformaldehyde (pH 6.0). Next, yeast cells were washed at pH 6.0 and incubated at pH 7.4 or pH 6.0 for 10 minutes. Finally, yeast cells were incubated with biotinylated CD3 antigen at opposite pH (cells incubated at pH 6.0 in the previous step were incubated at pH 7.4, whereas cells incubated at pH 7.4 in the previous step were incubated at pH 6.0) for 10 minutes. Binders labeled with biotinylated CD3 antigen were then sorted and characterized.

[0189] Three libraries from the initial MACS / FACS selection were obtained through four rounds of FACS selection. Approximately 1 × 10 8Yeast pellets were washed three times with wash buffer and incubated with 100 nM biotinylated CD3 antigen alone at room temperature for at least 10 minutes at pH 6.0, pH 7.4, or treated by pre-saturation and pH switching of method #2 discussed above. The yeast were then washed twice and stained with 1:100 dilution of goat anti-human F(ab')2κ-FITC (SouthernBiotech, Birmingham, Alabama, catalog number 2062-02), and 1:500 dilution of streptavidin-AlexaFluor 633 (Life Technologies, Grand Island, NY, catalog number S21375) or 1:50 dilution of Extravidin-phycoerthyrin (Sigma-Aldrich, St Louis, catalog number E4011), secondary reagents at 4°C for 15 minutes. After washing twice with ice-cold wash buffer, the cell pellet was resuspended in 0.4 mL wash buffer and transferred to a sorting tube with a filter cap. Sorting was performed using a FACS ARIA sorter (BD Biosciences) and sorting gates were determined to select for CD3 binders at pH 6 or non-binders at pH 7.4. Selected populations from the first round of FACS were carried forward to the next round.

[0190] The second, third, and fourth rounds of FACS for the selected populations above involved positive sorting for CD3 binders at pH 6.0 and negative sorting for reduced pH 7.4 binders and multispecific agent binders (Xu et al., Protein Eng Des Sel. 2013 Oct; 26(10): 663-70). In the second round of FACS (R3), cells were processed by pre-saturation and pH switching of method #2 (discussed above) or negatively sorted to selected non-binders at pH 7.4. In the third round of FACS (R4), the output from R3 was combined with the output from CD3 pre-saturation method #2 from R2 and subjected to CD3 pre-saturation method #1. In the final round of FACS (R5), the output from R4 was examined for PSR reactivity and human and cynomolgus monkey (Cyno) CD3 binding at pH 6 and 7.4. The output of each round was plated and isolates were selected for sequencing and characterization.

[0191] Figure 2Shown are exemplary FACS graphs from rounds 1 and 2 of selection from one library. Similar binding curves were observed in all libraries. In brief, during round 1, cells were positively sorted using 100 nM human CD3 ε δ heterodimer (HuCD3-hd) at pH 6. During round 2, cells were positively sorted using 100 nM HuCD3-hd at pH 6.0, negatively sorted using 100 nM HuCD3-hd at pH 7.4, or pre-saturated using method #2 described above. Binding to cynomolgus CD3 (CyCD3-hd) at pH 6.0 was also confirmed. Arrows indicate sorted cells that advance to the next round of sorting.

[0192] Figure 3 An exemplary FACS plot from round 3 is shown and compares the input from round 2 pH 6.0 positive sort and pH 7.4 negative sort. Briefly, sorts from round 2 were incubated with 100 nM HuCD3-hd at pH 6.0 and 7.4. The overlay column shows that the input cell population (from round 2 sort) exhibited higher binding at pH 6.0 compared to pH 7.4. Presaturation / Switch Method #2 was used to advance cells to the next round of selection.

[0193] Figure 4 Exemplary FACS plots from rounds 4 and 5 are shown. Round 4 compared cells incubated in 100 nM HuCD3-hd at pH 6 and pH 7.4. Round 4 also compared cells subjected to the pre-saturation / switch method at pH 6 and pH 7.4. Round 5 compared cells incubated in 100 nM HuCD3-hd or 100 nM CyCD3-hd at pH 6 (red) and pH 7.4 (grey).

[0194] Example 2: Determining the affinity of anti-CD3 antibodies to CD3

[0195] The kinetic constants (k a , k d , K D) was used to determine the affinity of anti-CD3 antibodies for CD3 at pH 6.0 and 7.4. ForteBio affinity measurements were generally performed as previously described (Estep et al., MAbs. 20135(2): 270-8). Briefly, ForteBio affinity measurements were performed by loading the antibody (IgG) onto the AHC sensor online. The sensor was equilibrated offline in the assay buffer for 30 minutes and then monitored online for 60 seconds to establish a baseline. For affinity binding measurements, the sensors with loaded IgG were exposed to 100 nM antigen (human or cynomolgus monkey CD3) for 3 minutes, after which they were transferred to the assay buffer for 3 minutes for off-rate measurements. In the data analysis software provided by ForteBio, a 1:1 binding model was used to fit the kinetic data. Table 2 provides the kinetic constants for the selected clones. Table 3 provides the equilibrium dissociation constants (K) of human CD3 for the selected clones at pH 6.0 and 7.4. D ).

[0196] FACS cell binding assays were used to determine the specificity of anti-CD3 antibodies to human CD3+Jurkat cells compared to CHO-S cells at pH 6.0 and 7.4. In brief, CD3+ human Jurkat cells and CHO-S cells were thawed and washed with cold PBSF buffer pH 7.4 (PBS+0.1% BSA, pH 7.4). Approximately 200,000 cells were aliquoted into each well of a 96-well plate and precipitated by centrifugation (5 minutes at 500x g). Cells were washed with PBSF pH 7.4 or PBSF pH 6.0 (PBS+0.1% BSA, pH 6.0), and then resuspended in 100ul of PBSF pH 7.4 or PBSF pH 6.0 with IgG antibodies (100nM) produced in yeast. The mixture (cells+antibody) was incubated on ice for 20 minutes, then washed twice with PBSF pH 7.4 or PBSF pH 6.0. Cells were resuspended in 50ul propidium iodide (1:500 dilution) and anti-human IgG-RPE (1:100 dilution) prepared in PBSF pH 7.4 or PBSF pH 6.0, then incubated on ice for 20 minutes in the dark, and then cells were washed twice with PBSF pH 7.4 or PBSF pH 6.0. Combination was analyzed on FACS Canto II. For selected clones, the mean fluorescence intensity (MFI) at pH 6.0 and 7.4 is shown in Table 3.

[0197] Figure 5A Shown are the HuCD3 binding responses at pH 6 (x-axis) compared to the HuCD3 binding responses at pH 7.4 (y-axis) for 236 unique clones from round 2 / 3 sorting output. Figure 5B Shown are the KD values ​​of HuCD3 (x-axis) at pH 6 compared to HuCD3 (y-axis) at pH 7.4 for 236 unique clones from the 2 / 3 round sorting output. Blue circles represent 2 / 3 round clones obtained by pre-saturation / switch sorting at pH 6.0, yellow circles represent 2 / 3 round clones obtained by negative sorting at pH 7.4, and red circles represent parental clone ADI-26906. The results show that negative sorting at pH 7.4 in round 2 / 3 tends to produce more pH-selected binders, but with weaker pH 6.0 responses or affinities, which are designated as Group 2 binders. Positive selection and pre-saturation / switch sorting at pH 6.0 produce clones with a variety of selectivities but with higher responses / affinity, which are designated as Group 1 binders.

[0198] Group 1 binding agents may include, for example, ADI-48592 (Abl25), ADI-48595 (Abl78), ADI-48650 (Ab77), ADI-48652 (Ab81), ADI-48662 (Abl16), and ADI-48666 (Abl77). Group 2 binding agents may 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 (Ab 182), ADI-48643(Ab46), ADI-48624(Ab241), ADI-48632(Ab15), ADI-48635(Ab17) and ADI-48645(Ab49).

[0199] Figure 6 Exemplary kinetics from ForteBio experiments of four clones compared to the parental clone ADI-26906 are provided. The K for each clone was calculated at pH 7.4 and pH 6.0. D By changing the K D Divide by the K at pH 6.0 D To obtain the ratio K DThe examples demonstrate that some clones designated as Group 1 binders, such as SAD10318_P02_A05 (ADI-48595) and SAD10318_P02_C04 (ADI-48592), bind more strongly (have lower K values) at pH 6.0 compared to pH 7.4. D Some clones designated as Group 2 binders, such as SAD10318_P01_A03 (ADI-48587) and SAD10318_P01_E01 (ADI-48577), were non-binders at pH 7.4, but bound at pH 6.0. Figure 6 The column "Sequence" (SEQ ID NOs: 576-590, respectively, in order of appearance) highlights amino acid substitutions in the CDRH3, CDRL1, and CDRL3 regions that may contribute to differential binding. Table 2 provides additional kinetic and PSR data for selected clones. 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 relative to binding at pH 7.4), low PSR scores, and provide a range of affinities for CD3.

[0200] 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 antibodies comprising a CDRH3 binding domain comprising a consensus motif comprising the sequence AX1DX2YX3HX4FYDV, wherein X1 is R or H, wherein X2 is A or H, wherein X3 is G, H or P, and wherein 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 included this sequence motif: 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;SAD1 0319_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;SAD1 0319_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; SAD1 0319_P05_E04;SAD10319_P05_F01;SAD10319_P06_B10;SAD10319_P06_B11;SAD10319_ 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.;

[0201] In some embodiments, the present disclosure provides antibodies comprising a CDRH3 binding domain comprising a consensus motif comprising the sequence ARDX1YGX2X3X4YDX5, wherein X1 is A or H, wherein X2 is R or H, wherein X3 is H or Y, wherein X4 is F or H, and wherein 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 included this 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_ 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_D1 1; SAD10319_P04_F12; SAD10319_P04_H12; SAD10320_P01_E02; SAD10320_P02_C04; SAD10320_P02_C06; AD10319_P05_B03;SAD10319_P05_B04;SAD10319_P05_D01;SAD10319_P05_G02;SAD10319_P05_G03;SAD10319_P05_H06;SAD10319_P06_A07;SAD1 1_A08; LAD9963_P01_E06; LAD9964_P01_C07; and LAD9966_P01_A08.

[0202] In some embodiments, the present disclosure provides antibodies comprising a CDRH3 binding domain comprising a consensus motif comprising the sequence ARDAHX1X2YX3X4DX5, wherein X1 is G, E, or R, wherein X2 is R or H, wherein X3 is F or H, wherein X4 is Y or H, and wherein 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 included this 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; SAD10318_P04_H23; SAD10318_P04_H34; SAD10318_P04_H46; SAD10318_P04_H57; SAD10318_P04_H60; SAD10318_P04_H71; SAD10318_P04_H82; SAD10318_P04_B04; SAD10318_P02_D04; SAD10318_P02_D06; SAD10318_P03_F07; SAD10318_P04_H12; SAD10318_P04_H AD10319_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.

[0203] In some embodiments, the present disclosure provides antibodies comprising a CDRH3 binding domain comprising a consensus motif comprising the sequence ARDAX1HRX2FYDV, wherein X1 is H, Y, S, G, A, T, V, or R, and wherein 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 include 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; SAD10318_P04_A13; SAD10318_P04_B07; SAD10318_P04_C08; SAD10318_P04_D09; SAD10318_P04_A14; SAD10318_P04_A15; SAD10318_P04_B08; SAD10318_P04_E08; SAD10318_P04_D09; SAD10318_P04_A16; SAD10318_P04_A17; SAD10318_P04_A18; SAD10318_P04_A19; SAD10318_P04_B09; SAD10318_P04_A12; SAD10318_P04_A13 AD10319_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.

[0204] In some embodiments, the present disclosure provides antibodies comprising a CDRH3 binding domain comprising a consensus motif comprising the sequence ARDX1YHRYFYDX2, wherein X1 is H or A, and wherein 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 include this 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_E09; SAD10318_P03_B101; SAD10318_P03_B11; SAD10318_P03_D01; SAD10318_P03_B12; SAD10318_P03_D03; SAD10318_P03_D04; SAD10318_P03_D05; SAD10318_P03_E07; SAD10318_P03_B13; SAD10318_P03_B14; SAD10318_P03_D08; SAD10318_P03_D09; SAD10318_P03_D01 AD10318_P03_D08; SAD10318_P03_E08; SAD10318_P03_F08; SAD10318_P03_G08; SAD10318_P03_C09; SAD10318_P04_B11; SAD10319_P01_H01; and SAD10319_P04_E12.

[0205] In some embodiments, the present disclosure provides antibodies comprising a CDRH3 binding domain comprising a consensus motif comprising the sequence AX1DAYX2X3X4HX5DV, wherein X1 is R or H, wherein X2 is G or H, wherein X3 is H or R, wherein X4 is N, F or Y, and wherein 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 included this 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.

[0206] In some embodiments, the present disclosure provides antibodies comprising a CDRH3 binding domain comprising a consensus motif comprising the sequence ARDX1X2GRYFYDV, wherein X1 is M, Q or H, and wherein X2 is R or H (SEQ ID NO:7). In some embodiments, at least one of X1 and X2 is H. The following 7 clones include this sequence motif: SAD10318_P02_E04; SAD10318_P04_C11; SAD10318_P04_F12; SAD10319_P02_H05; SAD10320_P01_A03; SAD10320_P01_B03; and SAD10320_P02_E05.

[0207] In some embodiments, the present disclosure provides antibodies comprising a CDRH3 binding domain comprising a consensus motif comprising the sequence ARDX1X2X3RYFYDX4, wherein X1 is H or A, wherein X2 is T, Y or H, wherein X3 is G or H, and wherein 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 this sequence motif: 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;A DI-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-485 97;ADI-48576;ADI-57277;ADI-57279;ADI-57280;ADI-57281;ADI-48601;ADI-48577;ADI-57284;ADI-4 8604;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.

[0208] In some embodiments, the disclosure provides antibodies comprising a CDRH3 binding domain comprising a consensus motif comprising the sequence ARDX1XXXX4YFYDX5, wherein X1 is H or A, wherein X2 is T, Y or H, wherein X3 is G or H, X4 is H, R, V or I, and wherein 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 this consensus motif: ADI-48576; ADI-48577; ADI-48587; ADI-48592; ADI-48595; ADI-48635; ADI-48650; ADI-48652; ADI-48666; ADI-48643; and ADI-48645.

[0209] In some embodiments, the present disclosure provides an antibody comprising a CD3 binding domain CDRH3, wherein the CDRH3 binding domain comprises a consensus motif comprising the sequence AX1DX2XXXXXXXXXXXXXXXXXX, wherein X1 is R or H, wherein X2 is A, H, M or Q, wherein X3 is Y, H, S, G, A, T, V or R; wherein X4 is G, H, P, E or R; wherein X5 is H or R, wherein X6 is Y, N, F, H, D, E, S, L, M, I, G, A, Q or T; wherein X7 is F or H; wherein X8 is Y or H; and wherein 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 include this sequence motif: 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_P0 4_H10;SAD10318_P04_D11;SAD10319_P01_A01;SAD10319_P01_C01;SAD10319_P01_E01;SAD10319_P01_A02;SAD10319_P01_C 02;SAD10319_P01_F02;SAD10319_P01_H02;SAD10319_P01_B03;SAD10319_P01_C03;SAD10319_P01_D03;SAD10319_P01_F03;

[0210] SAD10319_P02_A04; SAD10319_P02_C04; SAD10319_P02_E04;

[0211] SAD10319_P02_F04; SAD10319_P02_A05; SAD10319_P02_B05;

[0212] SAD10319_P02_C05; SAD10319_P02_G05; SAD10319_P02_A06;

[0213] SAD10319_P02_B06;SAD10319_P02_C06;SAD10319_P02_D06;

[0214] SAD10319_P02_F06;SAD10319_P02_G06;SAD10319_P03_C07;

[0215] SAD10319_P03_H07;SAD10319_P03_D08;SAD10319_P03_E08;

[0216] SAD10319_P03_E09;SAD10319_P03_F09;SAD10319_P04_A10;

[0217] SAD10319_P04_G10;SAD10319_P04_E11;SAD10319_P04_F11;

[0218] SAD10319_P04_G11;SAD10319_P04_C12;SAD10319_P04_D12;

[0219] SAD10320_P01_B01;SAD10320_P01_D01;SAD10320_P01_E01;

[0220] SAD10320_P01_G01;SAD10320_P01_H01;SAD10320_P01_A02;

[0221] SAD10320_P01_F02;SAD10320_P01_G02;SAD10320_P01_H02;

[0222] SAD10320_P01_C03;SAD10320_P01_D03;SAD10320_P01_E03;

[0223] SAD10320_P01_F03;SAD10320_P01_G03;SAD10320_P02_A04;

[0224] SAD10320_P02_B04;SAD10320_P02_E04;SAD10320_P02_H04;

[0225] SAD10320_P02_A05;SAD10320_P02_B05;SAD10320_P02_C05;

[0226] SAD10320_P02_B06;SAD10320_P02_D06;SAD10320_P02_E06;

[0227] SAD10320_P03_B07;SAD10320_P03_H07;SAD10320_P03_C08;

[0228] SAD10320_P03_D08;SAD10320_P03_F08;SAD10320_P03_H08;

[0229] SAD10320_P03_A09;SAD10320_P03_C09;SAD10320_P03_D09;

[0230] SAD10320_P03_F09;SAD10320_P04_A10;SAD10320_P04_C10;

[0231] SAD10320_P04_D10;SAD10320_P04_E10;SAD10320_P04_G10;

[0232] SAD10320_P04_D11;SAD10320_P04_E11;SAD10320_P04_F11;

[0233] SAD10320_P04_H11;SAD10320_P04_A12;SAD10320_P04_D12;

[0234] SAD10320_P04_E12;SAD10320_P04_F12;SAD10319_P05_A01;

[0235] SAD10319_P05_A05;SAD10319_P05_B02;SAD10319_P05_C01;

[0236] SAD10319_P05_C03;SAD10319_P05_C05;SAD10319_P05_D02;

[0237] SAD10319_P05_D03;SAD10319_P05_D05;SAD10319_P05_E04;

[0238] SAD10319_P05_F01;SAD10319_P06_B10;SAD10319_P06_B11;

[0239] SAD10319_P06_C10;SAD10319_P06_C12;SAD10319_P06_E08;

[0240] SAD10319_P06_F07;SAD10319_P06_F10;SAD10319_P06_G09;

[0241] SAD10319_P06_H07;SAD10319_P06_H08;SAD10319_P06_H10;

[0242] LAD5224_P03_A01;SAD10318_P01_B01;SAD10318_P01_F01;

[0243] SAD10318_P02_B05;SAD10318_P02_F05;SAD10318_P02_G05;

[0244] SAD10318_P03_B07;SAD10318_P03_G07;SAD10318_P03_A08;

[0245] SAD10318_P03_A09;SAD10318_P04_E10;SAD10318_P04_E11;

[0246] SAD10318_P04_H11;SAD10319_P01_D01;SAD10319_P01_F01;

[0247] SAD10319_P01_G01;SAD10319_P01_D02;SAD10319_P01_E02;

[0248] SAD10319_P02_B04;SAD10319_P02_E05;SAD10319_P02_E06;

[0249] SAD10319_P02_H06;SAD10319_P03_G08;SAD10319_P03_B09;

[0250] SAD10319_P03_G09;SAD10319_P04_B10;SAD10319_P04_C11;

[0251] SAD10319_P04_D11;SAD10319_P04_F12;SAD10319_P04_H12;

[0252] 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-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-57 277;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.;

[0253] In some embodiments, the disclosure provides antibodies comprising a CDRH3 binding domain comprising a consensus motif comprising the sequence ARDAX1XXXFYDX5, wherein X1 is T, H, or Y, wherein X2 is G or H, wherein X3 is H or R, wherein X4 is V or Y, and wherein 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 6 clones comprise this consensus motif and are designated as Group 1 binders: ADI-48592, ADI-48595, ADI-48650, ADI-48652, ADI-48662, and ADI-48666.

[0254] In some embodiments, the present disclosure provides antibodies comprising a CDRH3 binding domain comprising a consensus motif comprising the sequence AX1DX2XXXX5XXXX7YDX8, wherein X1 is R or H, wherein X2 is H or A, wherein X3 is H or Y, wherein X4 is H, G or P, wherein X5 is R or H, wherein X6 is Y, I or V, wherein X7 is F or H, and wherein 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 included this consensus motif and were 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.

[0255] In some embodiments, the present disclosure provides antibodies comprising a CDRH2 binding domain comprising a consensus motif comprising the sequence WIDLENANTIYDAKFQG (SEQ ID NO: 9). The following 148 clones included 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_P 01_G02;SAD10318_P01_A03;SAD10318_P01_C03;SAD10318_P01_D03;SAD10318_P01_E03;SAD10318_P01_F03;SAD10318_P01_G03;SAD10 318_P01_H03;SAD10318_P02_B04;SAD10318_P02_C04;SAD10318_P02_D04;SAD10318_P02_E04;SAD10318_P02_G04;SAD10318_P02_H04;S AD10318_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; 0318_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;SA D10320_P03_C09; SAD10320_P03_D09; SAD10320_P03_F09; SAD10320_P04_A10; SAD10320_P0 4_C10;SAD10320_P04_D10;SAD10320_P04_E10;SAD10320_P04_F10;SAD10320_P04_G10;SAD1 0320_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;SAD103 1_F02; 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. In addition, at least the following 16 clones included this consensus motif and were 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.;

[0256] In some embodiments, the present disclosure provides antibodies comprising a CDRH2 binding domain comprising a consensus motif comprising the sequence X1INPX2TGX3TX4YSQKFQG, wherein X1 is W or Y, wherein X2 is A, S, D, G, N, L, V, H or Q, wherein X3 is A, T or S, and wherein 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 included this consensus motif: SAD10319_P01_E02; SAD10319_P01_H02; SAD10319_P01_B03; SAD10319_P02_A04SAD10319_P02_B04; SAD10319_P02_C04; SAD10319_P02_F04; SAD10319_P02_H04; SAD10319_P02_A05; SAD10319_P02_C05; SAD10319_P02_C06; SAD10319_P 02_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.

[0257] In some embodiments, the present disclosure provides antibodies comprising a CDRH2 binding domain comprising a consensus motif comprising the sequence X1IX2AGTGX3TX4YSQKFQG, wherein X1 is W, Y or F, wherein X2 is T, N or D, wherein X3 is A, T or L, and wherein 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 included 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_A11; SAD10319_P04_B01; SAD10319_P04_A12; SAD10319_P04_B01; SAD10319_P04_A13; SAD10319_P04_A14; SAD10319_P04_A15; SAD10319_P04_A16; SAD10319_P04_A17; SAD10319_P04_A18; SAD10319_P04_A19; SAD10319_P04_A19; SAD10319_P04_A19; SAD10319_P04_A10; SAD10319_P04_A10; SAD10319_P04_A11; SAD10319_P04_A12; SAD10319_P04_A13; AD10319_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.

[0258] In some embodiments, the present disclosure provides antibodies comprising a CDRH2 binding domain comprising a consensus motif comprising the sequence X1IDAGTGX2TX3YSQKFQG, wherein X1 is S or W, wherein X2 is L, N, D or F, and wherein 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_P02_H07; SAD10319_P02_H08; SAD10319_P02_H09; SAD10319_P02_D01; SAD10319_P02_D02; SAD10319_P02_F03; SAD10319_P02_H06; SAD10319_P02_H07; SAD10319_P02_H08; SAD10319_P02_H09; SAD10319_P02_H09; SAD10319_P02_H01 AD10319_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.

[0259] In some embodiments, the present disclosure provides antibodies comprising a CDRH2 binding domain comprising a consensus motif comprising the sequence X1IX2AGTGATX3YSQKFQG, wherein X1 is G, D or S, wherein X2 is I or D, and wherein 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.

[0260] In some embodiments, the present disclosure provides antibodies comprising a CDRH2 binding domain comprising a consensus motif comprising the sequence WINPX1TGNTX2YSQKFQG, wherein X1 is D, T, L, S, or A, and wherein 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 6 clones include this consensus motif: SAD10319_P01_A01; SAD10319_P01_F01; SAD10319_P01_C02; SAD10319_P04_F12; SAD10319_P05_E04; and SAD10319_P06_A11.

[0261] In some embodiments, the present disclosure provides antibodies comprising a CDRH2 binding domain comprising a consensus motif comprising the sequence X1INAGTGX2TX3YSQKFQG, wherein X1 is Y or W, wherein X2 is N, D or A, and wherein X3 is I or V (SEQ ID NO: 15). In some embodiments, at least one of X1, X2 and X3 is H. The following 5 clones include this consensus motif: SAD10319_P01_F03; SAD10319_P02_H05; SAD10319_P02_D06; SAD10319_P03_E08; and SAD10319_P03_H08.

[0262] In some embodiments, the present disclosure provides antibodies comprising a CDRH2 binding domain comprising a consensus motif comprising the sequence X1INPX2TGX3TKYSQKFQG, wherein X1 is W or Y, wherein X2 is D, I or Y, and wherein 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 5 clones include this consensus motif: SAD10319_P03_H07; SAD10319_P04_E11; SAD10319_P04_F11; SAD10319_P04_B12; and SAD10319_P04_D12.

[0263] In some embodiments, the present disclosure provides antibodies comprising a CDRH2 binding domain comprising a consensus motif comprising the sequence SIX1AGTGX2TKYSQKFQG, wherein X1 is N or V, and wherein X2 is A or I (SEQ ID NO: 17). In some embodiments, at least one of X1 and X2 is H. The following 3 clones include this consensus motif: SAD10319_P02_E04; SAD10319_P04_C10; and SAD10319_P04_H12.

[0264] In some embodiments, the present disclosure provides antibodies comprising a CDRH2 binding domain comprising a consensus motif comprising the sequence SINAGTGX1TX2YSQKFQG, wherein X1 is F or N, and wherein X2 is Y or D (SEQ ID NO: 18). In some embodiments, at least one of X1 and X2 is H. The following 3 clones include this consensus motif: SAD10319_P02_B05; SAD10319_P02_B06; and SAD10319_P05_D02.

[0265] In some embodiments, the present disclosure provides antibodies comprising a CDRH2 binding domain comprising a consensus motif comprising the sequence X1IX2X3GTGX4TDYSQKFQG, wherein X1 is D or W, wherein X2 is N or H, wherein X3 is A or S, and wherein 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 3 clones include this consensus motif: SAD10319_P05_B03; SAD10319_P05_B04; and SAD10319_P05_D04.

[0266] In some embodiments, the present disclosure provides antibodies comprising a CDRH2 binding domain comprising a consensus motif comprising the sequence WIDPX1TGATX2YSQKFQG, wherein X1 is N, H or Y, and wherein X2 is V or K (SEQ ID NO: 20). In some embodiments, at least one of X1 and X2 is H. The following 3 clones include this consensus motif: SAD10319_P01_C03; SAD10319_P03_G09; and SAD10319_P06_F10.

[0267] In some embodiments, the present disclosure provides antibodies comprising a CDRH2 binding domain comprising a consensus motif comprising the sequence WIX1PX2TGNTKYSQKFQG, wherein X1 is D or N, and wherein X2 is L, I or V (SEQ ID NO: 21). In some embodiments, at least one of X1 and X2 is H. The following 3 clones include this consensus motif: SAD10319_P01_A02; SAD10319_P04_C12; and SAD10319_P05_G02.

[0268] In some embodiments, the present disclosure provides antibodies comprising a CDRH2 binding domain comprising a consensus motif comprising the sequence SINAGDANTKYSQKFQG (SEQ ID NO: 22). The following 2 clones include this consensus motif: SAD10319_P04_G11 and SAD10319_P06_H07.

[0269] In some embodiments, the present disclosure provides antibodies comprising a CDRH2 binding domain comprising a consensus motif comprising the sequence X1IDPX2TGATX3YSQKFQG, wherein X1 is D or W, wherein X2 is D or V, and wherein X3 is E or D (SEQ ID NO: 23). In some embodiments, at least one of X1, X2, and X3 is H. The following 2 clones include this consensus motif: SAD10319_P05_G04 and SAD10319_P06_E08.

[0270] In some embodiments, the disclosure provides antibodies comprising a CDRH2 binding domain comprising a consensus motif comprising the sequence WINAGDAATVYSQKFQG (SEQ ID NO: 24). The following 2 clones include this consensus motif: SAD10319_P06_G11 and SAD10319_P06_H11.

[0271] In some embodiments, the present disclosure provides antibodies comprising a CDRH2 binding domain comprising a consensus motif comprising the sequence XI IX2XXXXXXTX8YSQKFQG, wherein X1 is W, S, Y, F, G, or D, wherein X2 is N, T, D, V, or H, wherein X3 is A, P, or S, wherein X4 is G, A, S, N, D, L, V, H, Q, T, I, or Y, wherein X5 is D or T, wherein X6 is A or G, wherein X7 is A, N, T, S, L, D, F, Y, or E, and wherein 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 included this consensus motif: SAD10319_P01_E02; SAD10319_P01_H02; SAD10319_P01_B03; SAD10319_P02_A04SAD10319_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; SAD10 319_P03_F09;SAD10319_P04_G10;SAD10319_P04_C11;SAD10319_P05_A01;SAD10319_P05_A05;SAD10319_P0 5_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; SAD10 319_P03_G07;SAD10319_P03_B09;SAD10319_P03_E09;SAD10319_P04_A10;SAD10319_P04_B10;SAD10319_P0 4_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.;

[0272] In some embodiments, the disclosure provides antibodies comprising a CDRH2 binding domain comprising a consensus motif comprising the sequence WIDAGTGX1TX2YSQKFQG, wherein X1 is L, F, N or A, and wherein X2 is T or K (SEQ ID NO: 595). At least the following 4 clones included this consensus motif and were designated as Group 2 binders: ADI-48636, ADI-48638, ADI-48624, and ADI-48635.

[0273] In some embodiments, the present disclosure provides antibodies comprising a CDRH1 binding domain comprising a consensus motif comprising the sequence X1NIKDYX2MH, wherein X1 is F or S, and wherein 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 included 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_D 02;SAD10318_P01_F02;SAD10318_P01_G02;SAD10318_P01_A03;SAD10318_P01_C03;SAD10318_P01_D03;SAD10318_P0 1_E03;SAD10318_P01_F03;SAD10318_P01_G03;SAD10318_P01_H03;SAD10318_P02_B04;SAD10318_P02_C04;SAD10318_ P02_D04;SAD10318_P02_E04;SAD10318_P02_G04;SAD10318_P02_H04;SAD10318_P02_A05;SAD10318_P02_B05;SAD103 18_P02_C05;SAD10318_P02_D05;SAD10318_P02_F05;SAD10318_P02_G05;SAD10318_P02_H05;SAD10318_P02_B06;SAD1 0318_P02_D06;SAD10318_P02_E06;SAD10318_P02_G06;SAD10318_P03_B07;SAD10318_P03_E07;SAD10318_P03_F07;SA D10318_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; SAD1032 0_P02_F06;SAD10320_P02_G06;SAD10320_P02_H06;SAD10320_P03_B07;SAD10320_P03_E07;SAD10320_P03_H0 7;SAD10320_P03_C08;SAD10320_P03_D08;SAD10320_P03_F08;SAD10320_P03_H08;SAD10320_P03_A09;SAD10 320_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;SAD 10320_P04_E11; SAD10320_P04_F11; SAD10320_P04_G11; SAD10320_P04_H11; SAD10320_P04_A12; SAD10320_P0 4_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.;

[0274] In some embodiments, the present disclosure provides antibodies comprising a CDRH1 binding domain comprising a consensus motif comprising the sequence YTFX1XXXMH, wherein X1 is A, K, D, Q, E, N, T, L, Y, S, P, G, H, or V, wherein X2 is T, S, or A, wherein X3 is Y or I, and wherein 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 included 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_D 04;SAD10319_P02_E04;SAD10319_P02_F04;SAD10319_P02_H04;SAD10319_P02_A05;SAD10319_P02_E05;SAD10319_P0 2_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;SAD103 19_P03_E09;SAD10319_P03_F09;SAD10319_P03_G09;SAD10319_P04_B11;SAD10319_P04_C11;SAD10319_P04_D11;SAD1 0319_P04_G11;SAD10319_P04_B12;SAD10319_P04_C12;SAD10319_P04_D12;SAD10319_P04_E12;SAD10319_P05_A01;SA D10319_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.;

[0275] In some embodiments, the present disclosure provides antibodies comprising a CDRH1 binding domain comprising a consensus motif comprising the sequence YTFX1X2X3X4MH, wherein X1 is T, D, A, N, or V, wherein X2 is D, E, G, or Q, wherein X3 is Y or D, and wherein 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 included 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; S AD10319_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;S AD10319_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;S AD10319_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.

[0276] In some embodiments, the present disclosure provides antibodies comprising a CDRH1 binding domain comprising a consensus motif comprising the sequence YTFTSX1X2MH, wherein X1 is A, D or T, and wherein 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 7 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.

[0277] In some embodiments, the present disclosure provides antibodies comprising a CDRH1 binding domain comprising a consensus motif comprising the sequence YTFX1X2YX3MH, wherein 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 4 clones include this consensus motif: SAD10319_P02_F05; SAD10319_P02_G05; SAD10319_P02_H05; and SAD10319_P05_G01.

[0278] In some embodiments, the present disclosure provides antibodies comprising a CDRH1 binding domain comprising a consensus motif comprising the sequence YTFX1X2YVMH, wherein X1 is I or N, and wherein X2 is K or R (SEQ ID NO: 32). In some embodiments, at least one of X1 and X2 is H. The following 2 clones include this consensus motif: SAD10319_P06_D12 and SAD10319_P06_E08.

[0279] In some embodiments, the disclosure provides antibodies comprising a CDRH1 binding domain comprising a consensus motif comprising the sequence FNIKDYYMH (SEQ ID NO: 47). At least the following 6 clones included this consensus motif and were designated as Group 1 binders: ADI-48592, ADI-48595, ADI-48650, ADI-48652, ADI-48662, and ADI-48666. In addition, at least the following 16 clones included this consensus motif and were 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.

[0280] In some embodiments, the disclosure provides antibodies comprising a CDRH1 binding domain comprising a consensus motif comprising the sequence YTFX1X2YX3MH, wherein X1 is E, S or T, wherein X2 is S or D, and wherein X3 is A or D (SEQ ID NO: 31). At least the following 5 clones included this consensus motif and were designated as Group 2 binders: ADI-48636, ADI-48638, ADI-48624, ADI-48632, and ADI-48635.

[0281] In some embodiments, the present disclosure provides antibodies comprising a CDRL3 binding domain comprising a consensus motif comprising the sequence X1X2SX3X4X5RX6, wherein X1 is H, K or G, wherein X2 is Q or H, wherein X3 is Y or H, wherein X4 is S, H, D, T, V, M or L, wherein X5 is R or H, and wherein 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 included this 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_B03; SAD10318_P01_D03; 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; S AD10318_P01_C03;SAD10318_P01_D03;SAD10318_P01_E03;SAD10318_P01_F03;SAD10318_P02_B04;SAD10318_P02_C04;S AD10318_P02_D04;SAD10318_P02_E04;SAD10318_P02_H04;SAD10318_P02_A05;SAD10318_P02_B05;SAD10318_P02_C05;S AD10318_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_B05SAD10319_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; SAD10 319_P04_D11;SAD10319_P04_E11;SAD10319_P04_F11;SAD10319_P04_G11;SAD10319_P04_B12;SAD10319_P04_C12;SAD10319_P0 4_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;SAD10 320_P02_B05;SAD10320_P02_D05;SAD10320_P02_H05;SAD10320_P02_A06;SAD10320_P02_E06;SAD10320_P02_F06;SAD10320_P 02_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.;

[0282] In some embodiments, the present disclosure provides antibodies comprising a CDRL3 binding domain comprising a consensus motif comprising the sequence KQSYX1X2RT, wherein X1 is H, V, K, W, R, L, G, Y, or Q, and wherein 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 included 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_P 03_C09;SAD10318_P03_E09;SAD10318_P04_A12;SAD10320_P01_B01;SAD10320_P01_E01;SAD10320_P01_G01;SAD10 320_P01_A02;SAD10320_P01_F02;SAD10320_P01_G02;SAD10320_P01_C03;SAD10320_P01_D03;SAD10320_P01_E03;S AD10320_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_ SAD10320_P04_E11; SAD10320_P04_G11; SAD10320_P04_H11; SAD10320_P04_A12; and SAD10320_P04_E12.

[0283] In some embodiments, the present disclosure provides antibodies comprising a CDRL3 binding domain comprising a consensus motif comprising the sequence X1QSX2HX3RT, wherein X1 is K or H, wherein X2 is H, Y, M, S, L, E, G or W, and wherein 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 included 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.

[0284] In some embodiments, the present disclosure provides antibodies comprising a CDRL3 binding domain comprising a consensus motif comprising the sequence KQSX1X2X3RT, wherein 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 include 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.

[0285] In some embodiments, the disclosure provides antibodies comprising a CDRL3 binding domain comprising a consensus motif comprising the sequence KQSX1X2X3RT, wherein X1 is H or Y, wherein X2 is T, S or Q, and wherein 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 comprised this consensus motif and were designated as Group 1 binders: ADI-48592, ADI-48595, ADI-48650, ADI-48652, ADI-48662, and ADI-48666.

[0286] In some embodiments, the present disclosure provides antibodies comprising a CDRL3 binding domain comprising a consensus motif comprising the sequence X1QSX2XXXRT, wherein X1 is K or H, wherein X2 is Y or H, wherein X3 is S, H, L, V or K, and wherein 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 included this consensus motif and were 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.

[0287] In some embodiments, the present disclosure provides antibodies comprising a CDRL2 binding domain comprising a consensus motif comprising the sequence WASTRES (SEQ ID NO: 37). The following 215 clones included 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_P 01_G02;SAD10318_P01_A03;SAD10318_P01_C03;SAD10318_P01_D03;SAD10318_P01_E03;SAD10318_P01_F03;SAD10318_P01_G03;SAD10 318_P01_H03;SAD10318_P02_B04;SAD10318_P02_C04;SAD10318_P02_D04;SAD10318_P02_E04;SAD10318_P02_G04;SAD10318_P02_H04;S AD10318_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; 0318_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; SAD103 20_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;S AD10320_P03_D09; SAD10320_P03_F09; SAD10320_P04_A10; SAD10320_P04_C10; SAD10320_P04_D10; SAD10320_P04_E10; SAD1032 0_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;LAD In addition, at least the following 6 clones included this consensus motif and were 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 included this consensus motif and were 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.;

[0288] In some embodiments, the present disclosure provides antibodies comprising a CDRL1 binding domain comprising a consensus motif comprising the sequence KSSQSLLX1X2X3X4GX5NX6LA, wherein X1 is N or H, wherein X2 is A, R or T, wherein X3 is R or H, wherein X4 is T, P or E, wherein X5 is H or K, and wherein 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 included 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_P 01_D02;SAD10318_P01_F02;SAD10318_P01_G02;SAD10318_P01_A03;SAD10318_P01_C03;SAD10 318_P01_D03;SAD10318_P01_E03;SAD10318_P01_F03;SAD10318_P01_G03;SAD10318_P01_H03;S AD10318_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;SAD1 0318_P02_G06;SAD10318_P03_B07;SAD10318_P03_E07;SAD10318_P03_F07;SAD10318_P03_G07;

[0289] SAD10318_P03_A08; SAD10318_P03_B08; SAD10318_P03_C08;

[0290] SAD10318_P03_D08; SAD10318_P03_E08; SAD10318_P03_F08;

[0291] SAD10318_P03_G08;SAD10318_P03_H08;SAD10318_P03_A09;

[0292] SAD10318_P03_C09;SAD10318_P03_D09;SAD10318_P03_E09;

[0293] SAD10318_P03_F09;SAD10318_P03_G09;SAD10318_P04_C10;

[0294] SAD10318_P04_D10;SAD10318_P04_E10;SAD10318_P04_H10;

[0295] SAD10318_P04_A11;SAD10318_P04_B11;SAD10318_P04_C11;

[0296] SAD10318_P04_D11;SAD10318_P04_E11;SAD10318_P04_F11;

[0297] SAD10318_P04_G11;SAD10318_P04_H11;SAD10318_P04_A12;

[0298] SAD10318_P04_F12;SAD10318_P04_G12;SAD10318_P04_H12;

[0299] SAD10319_P01_A01;SAD10319_P01_C01;SAD10319_P01_D01;

[0300] SAD10319_P01_E01;SAD10319_P01_F01;SAD10319_P01_G01;

[0301] SAD10319_P01_H01;SAD10319_P01_A02;SAD10319_P01_C02;

[0302] SAD10319_P01_D02;SAD10319_P01_E02;SAD10319_P01_F02;

[0303] SAD10319_P01_H02;SAD10319_P01_B03;SAD10319_P01_C03;

[0304] SAD10319_P01_D03;SAD10319_P01_F03;SAD10319_P02_A04;

[0305] SAD10319_P02_B04;SAD10319_P02_C04;SAD10319_P02_D04;

[0306] SAD10319_P02_E04;SAD10319_P02_F04;SAD10319_P02_H04;

[0307] SAD10319_P02_A05;SAD10319_P02_B05;SAD10319_P02_C05;

[0308] SAD10319_P02_D05;SAD10319_P02_E05;SAD10319_P02_F05;

[0309] SAD10319_P02_G05;SAD10319_P02_H05;SAD10319_P02_A06;

[0310] SAD10319_P02_B06;SAD10319_P02_C06;SAD10319_P02_D06;

[0311] SAD10319_P02_E06;SAD10319_P02_F06;SAD10319_P02_G06;

[0312] SAD10319_P02_H06;SAD10319_P03_C07;SAD10319_P03_G07;

[0313] SAD10319_P03_H07;SAD10319_P03_D08;SAD10319_P03_E08;

[0314] SAD10319_P03_G08;SAD10319_P03_H08;SAD10319_P03_B09;

[0315] SAD10319_P03_E09;SAD10319_P03_F09;SAD10319_P03_G09;

[0316] SAD10319_P04_A10;SAD10319_P04_B10;SAD10319_P04_C10;

[0317] SAD10319_P04_G10;SAD10319_P04_B11;SAD10319_P04_C11;

[0318] SAD10319_P04_D11;SAD10319_P04_E11;SAD10319_P04_F11;

[0319] SAD10319_P04_G11;SAD10319_P04_B12;SAD10319_P04_C12;

[0320] SAD10319_P04_D12;SAD10319_P04_E12;SAD10319_P04_F12;

[0321] SAD10319_P04_H12;SAD10320_P01_B01;SAD10320_P01_D01;

[0322] SAD10320_P01_E01;SAD10320_P01_F01;SAD10320_P01_G01;

[0323] SAD10320_P01_H01;SAD10320_P01_A02;SAD10320_P01_C02;

[0324] SAD10320_P01_E02;SAD10320_P01_F02;SAD10320_P01_G02;

[0325] SAD10320_P01_H02;SAD10320_P01_A03;SAD10320_P01_B03;

[0326] SAD10320_P01_C03;SAD10320_P01_D03;SAD10320_P01_E03;

[0327] SAD10320_P01_F03;SAD10320_P01_G03;SAD10320_P01_H03;

[0328] SAD10320_P02_A04;SAD10320_P02_B04;SAD10320_P02_C04;

[0329] SAD10320_P02_E04;SAD10320_P02_H04;SAD10320_P02_A05;

[0330] SAD10320_P02_B05; SAD10320_P02_C05; SAD10320_P02_D05;

[0331] 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_D0 8;SAD10320_P03_F08;SAD10320_P03_A09;SAD10320_P03_C09;SAD10320_P03_D09;SAD10320_P03_F0 9;SAD10320_P04_A10;SAD10320_P04_C10;SAD10320_P04_D10;SAD10320_P04_E10;SAD10320_P04_F 10;SAD10320_P04_G10;SAD10320_P04_A11;SAD10320_P04_D11;SAD10320_P04_E11;SAD10320_P04_F 11; 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.

[0332] In some embodiments, the present disclosure provides antibodies comprising a CDRL1 binding domain comprising a consensus motif comprising the sequence KSSQSLLX1AX2THX3NX4LA, wherein X1 is N or H, wherein X2 is R or H, wherein X3 is K or H, and wherein 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 included 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.

[0333] In some embodiments, the present disclosure provides antibodies comprising a CDRL1 binding domain comprising a consensus motif comprising the sequence KSSQSLLNASTAKNYLA (SEQ ID NO: 40) or KSSQSLLNARTRTNYLA (SEQ ID NO: 41).

[0334] In some embodiments, the present disclosure provides antibodies comprising a CDRL1 binding domain comprising a consensus motif comprising the sequence KSSQSLLNX1X2X3GX4NX5LA, wherein 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 include 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.

[0335] In some embodiments, the disclosure provides antibodies comprising a CDRL1 binding domain comprising a consensus motif comprising the sequence KSSQSLLNX1X2TGX3NYLA, wherein X1 is A or S, wherein X2 is R or H, and wherein 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 6 clones included this consensus motif and were designated as Group 1 binders: ADI-48592, ADI-48595, ADI-48650, ADI-48652, ADI-48662, and ADI-48666.

[0336] In some embodiments, the present disclosure provides antibodies comprising a CDRL1 binding domain comprising a consensus motif comprising the sequence KSSQSLLX1AX2X3X4X5NX6LA, wherein X1 is N or H, wherein X2 is R or H, wherein X3 is T or E, wherein X4 is G or H, wherein X5 is H or K, and wherein X6 is H or Y (SEQ ID NO: 597). In some embodiments, at least one of X1, X2, X4, X5, and X6 is H. At least the following 21 clones included this consensus motif and were 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.

[0337] In some embodiments, the disclosure provides antibodies comprising a CDRH3 binding domain comprising a consensus motif comprising the sequence AX1DX2YX3HX4FYDV, wherein X1 is R or H, wherein X2 is A or H, wherein X3 is G, H or P, and wherein 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 comprising the sequence WIDLENANTIYDAKFQG (SEQ ID NO: 9); WINPX1TGATX2YSQKFQG, wherein X1 is S, D, A, N, L, or Q, and wherein X2 is V, T, D, Y, or K (SEQ ID NO: 45); or XIDAGTGATX2YSQKFQG, wherein X1 is W, S, or D, and wherein X2 is A, H, K, T, or D (SEQ ID NO: 50). NO:46); a CDRH1 binding domain comprising a consensus motif comprising the sequence FNIKDYYMH (SEQ ID NO:47); a CDRL3 binding domain comprising a consensus motif comprising the sequence KSQYX1X2RT, wherein X1 is S, H, V, K, W, L, G, T, R, or Q, and wherein 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 comprising the sequence WASTRES (SEQ ID NO:37), and a CDRL1 binding domain comprising a consensus motif comprising the sequence KSSQSLLNARTGKNYLA (SEQ ID NO:49).

[0338] In some embodiments, the disclosure provides antibodies comprising a CDRH3 binding domain comprising a consensus motif comprising the sequence ARDX1YGX2X3X4YDX5, wherein X1 is A or H, wherein X2 is R or H, wherein X3 is H or Y, wherein X4 is F or H, and wherein X5 is H or V (SEQ ID NO: 2); a CDRH2 binding domain comprising a consensus motif comprising the sequence WIDLENANTIYDAKFQG (SEQ ID NO: 9) or WIX1AGTGATX2YSQKGQG, wherein X1 is T, N or D, and wherein X2 is V or K (SEQ ID NO: 50); a CDRH1 binding domain comprising a consensus motif comprising the sequence FNIKDYYMH (SEQ ID NO:47) or YTFX1X2YX3MH, wherein X1 is T or A, X2 is E, D, A, S, G or Q, and X3 is D, A, V or E (SEQ ID NO:51); a CDRL3 binding domain containing a consensus motif comprising the sequence KQSX1SRRT, wherein X1 is H or Y (SEQ ID NO:52); a CDRL2 binding domain containing a consensus motif comprising the sequence WASTRES (SEQ ID NO:37); and a CDRL1 binding domain containing a consensus motif comprising the sequence KSSQSLLX1AX2TX3X4NX5LA, wherein 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).

[0339] In some embodiments, the present disclosure provides antibodies comprising a CDRH3 binding domain comprising a consensus motif comprising the sequence ARDAHX1X2YX3X4DX5, wherein X1 is G, E, or R, wherein X2 is R or H, wherein X3 is F or H, wherein X4 is Y or H, and wherein X5 is V or H (SEQ ID NO: 3); a CDRH2 binding domain comprising a consensus motif comprising the sequence WIDLENANTIYDAKFQG (SEQ ID NO: 9); a CDRH1 binding domain comprising a consensus motif comprising the sequence FNIKDYYMH (SEQ ID NO: 47); a CDRL3 binding domain comprising a consensus motif comprising the sequence KQSYSRRT (SEQ ID NO: 54); a CDRL2 binding domain comprising a consensus motif comprising the sequence WASTRES (SEQ ID NO: 103); NO:37); and a CDRL1 binding domain containing a consensus motif comprising the sequence KSSQSLLNAX1TGX2NX3LA, wherein X1 is H or R, wherein X2 is H or K, and X3 is H or Y (SEQ ID NO:55).

[0340] In some embodiments, the present disclosure provides antibodies or antigen-binding fragments comprising a CDRH3 binding domain comprising a consensus motif comprising the sequence ARDAX1X2X3X4FYDX5, wherein X1 is T, H or Y, wherein X2 is G or H, wherein X3 is H or R, wherein X4 is V or Y, wherein X5 is V or H, and wherein, optionally, at least one of X1, X2, X3 and X5 is H (SEQ ID NO: 593); a CDRH2 binding domain comprising a consensus motif comprising the sequence WIDLENANTIYDAKFQG (SEQ ID NO: 9); a CDRH1 binding domain comprising a consensus motif comprising the sequence FNIKDYYMH (SEQ ID NO:47); a CDRL3 binding domain comprising a consensus motif comprising the sequence KQSX1X2X3RT, wherein X1 is H or Y, wherein X2 is T, S or Q, wherein X3 is R or H, and optionally, wherein at least one of X1 and X3 is H (SEQ ID NO:36); a CDRL2 binding domain comprising a consensus motif comprising the sequence WASTRES (SEQ ID NO:37); and / or a CDRL1 binding domain comprising a consensus motif comprising the sequence KSSQSLLNX1X2TGX3NYLA, wherein X1 is A or S, wherein X2 is R or H, wherein X3 is H or K, and optionally, wherein 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 binding agent comprising a CD3 binding domain selected from ADI-48592, ADI-48595, ADI-48650, ADI-48652, ADI-48662, and ADI-48666.

[0341] In some embodiments, the present disclosure provides antibodies or antigen-binding fragments comprising a CDRH3 binding domain comprising a consensus motif comprising the sequence AX1DX2XXXX5X6X7YDX8, wherein X1 is R or H, wherein X2 is H or A, wherein X3 is H or Y, wherein X4 is H, G or P, wherein X5 is R or H, wherein X6 is Y, I or V, wherein X7 is F or H, wherein X8 is V or H, and optionally, wherein 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 comprising the sequence WIDLENANTIYDAKFQG (SEQ ID NO: 9) or the sequence WIDAGTGX1TX2YSQKFQG, wherein X1 is L, F, N or A, and wherein X2 is T or K (SEQ ID NO: 103); NO:595); a CDRH1 binding domain comprising a consensus motif comprising the sequence FNIKDYYMH (SEQ ID NO:47) or the sequence YTFX1X2YX3MH, wherein X1 is E, S or T, wherein X2 is S or D, and wherein X3 is A or D (SEQ ID NO:31); a CDRL3 binding domain comprising a consensus motif comprising the sequence X1QSX2X3X4RT, wherein X1 is K or H, wherein X2 is Y or H, wherein X3 is S, H, L, V or K, wherein X4 is H, R or E, and optionally, wherein at least one of X1, X2, X3 and X4 is H (SEQ ID NO:598); a CDRL2 binding domain comprising a consensus motif comprising the sequence WASTRES (SEQ ID NO:37); and / or a CDRL1 binding domain containing a consensus motif comprising the sequence KSSQSLLX1AX2X3X4X5NX6LA, wherein X1 is N or H, wherein X2 is R or H, wherein X3 is T or E, wherein X4 is G or H, wherein X5 is H or K, wherein X6 is H or Y, and wherein, optionally, at least one of X1, X2, X4, X5 and X6 is H (SEQ ID NO:597).In some embodiments, the antibody or antigen-binding fragment is designated as a Group 2 binding agent 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.

[0342] Materials and methods

[0343] In addition to the description provided above, the following materials and methods were used in the examples.

[0344] Hu and Cy CD3Fc heterodimeric antigen production. Recombinant heterodimeric CD3Fc fusion antigens were produced in HEK293 cells by co-transfection of plasmids encoding Hu CD3Fc (extracellular domain, ECD, residues 22-126) and CD3Fc-HIS (ECD residues 22-100) or Cy CD3Fc (ECD residues 22-117) and CD3Fc-HIS (ECD residues 22-100) with heterologous signal peptide sequences. Chromatographic separation was performed on an Avant 150 preparative chromatography system (GE Healthcare Life Sciences) to enable online salt concentration monitoring during the run. The clarified culture supernatant was purified by Ni Sepharose 6 Fast Flow (GE Healthcare Life Sciences), which removed CD3Fc-HIS homodimers. CD3Fc-HIS heterodimers were separated from CD3Fc-HIS homodimers by a linear Tris-buffered KCl gradient at pH 8.5 through Mono Q 10 / 100GL.

[0345] Peptides. C-terminal biotinylated CD3 N-terminal peptides were obtained from New England Peptide. All peptides were delivered at a purity of ≥95%. Peptides were designed based on the primary sequence of Hu CD3 bound to OKT3 and the crystal structure of Hu CD3 (Kjer-Nielsen L. et al. PNAS 2004). The CD3N27 peptide has the sequence H2N-QDGNEEMGSITQTPYQVSISGTTVILT[K / SCBiot(dPEG4)]-amide (SEQ ID NO: 56) and the CD3N13 peptide has the sequence H2N-QDGNEEMGGITQT[K / SCBiot(dPEG4)]-amide (SEQ ID NO: 57).

[0346] Antigen biotinylation. CD3 antigen was biotinylated using the EZ-Link Sulfo-NHS-biotinylation kit from Pierce. Goat anti-human F(ab')2κ-FITC (LC-FITC), Extravidin-PE (EA-PE), and streptavidin 633 (SA-633) were obtained from Southern Biotech, Sigma, and Molecular Probes, respectively. Streptavidin microbeads and MACS LC separation columns were purchased from Miltenyi Biotec.

[0347] Cell line propagation and cell marker assays. 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 Nonhuman Primate Reagent Resource. All cell lines were cultured in RPMI 1640 GlutaMax medium supplemented with 10% fetal bovine serum (FBS).

[0348] Cell labeling is carried out by dividing 100,000-200,000 cells in each hole in a 96-well assay plate.Cells are centrifuged at 4C for 5 minutes with 500x g, then resuspended in 100nM IgG of 100l and incubated at room temperature for 20 minutes.Then cells are washed three times in buffer (phosphate buffered saline (PBS) / 0.1% bovine serum albumin (BSA)), and resuspended in secondary reagent, usually goat anti-human R-PE (Southern Biotech).Plate is measured on FACSCanto (BD Biosciences) using HTS sample injector.Flow cytometry data are analyzed for the median fluorescence intensity in R-PE channel.

[0349] FACS affinity pressure selection method. Briefly, yeast cells (at least about 2 x 107 Cells / labeling condition) are incubated with a volume of biotinylated antigen sufficient to represent a stoichiometric excess relative to the average IgG presentation number. Antigen labeling conditions are 100 to 1 nM under equilibrium conditions, usually in FACS wash buffer (phosphate buffered saline (PBS) / 0.1% bovine serum albumin (BSA)) for 20 minutes to several hours at room temperature. After washing three times with wash buffer, the yeast is then stained with a secondary reagent anti-human light chain FITC conjugate (LC-FITC) diluted 1:100, and streptavidin-633 (SA-633) diluted 1:500 or extravidin-phycoerythrin (EA-PE) diluted 1:50 at 4°C for 15 minutes. After washing twice with ice-cold wash buffer, the cell pellet is washed with at least 1 mL / 1x 10 7 A typical volume of 1000 yeast was resuspended in wash buffer and transferred to a sorting tube with a filter cap. Sorting was performed using a FACS ARIA sorter (BD Biosciences), and sorting gates were determined to select binders. After the last round of sorting, yeast plates were inoculated and single colonies were picked for characterization.

[0350] Antibody yeast production and purification. Yeast clones were grown to saturation and then induced with shaking at 30°C for 48 hours. After induction, yeast cells were precipitated and harvested supernatant for purification. Protein A column was used to purify IgG and eluted with acetic acid pH 2.0. Fab fragments were produced by papain digestion and purified on KappaSelect or CaptureSelect IgG-CH1 (GE Healthcare LifeSciences).

[0351] Antibody HEK production and purification. Mammalian expression of IgG is accomplished by subcloning the antibody into a new expression vector, followed by transient transfection and expression in HEK293ADI1 (a monoclonal cell line derived from HEK293 (DSMZ) selected for non-aggregating growth, growth rate and transfection). In brief, the expression vector containing the antibody of interest is transfected by complexing with a transfection reagent, then exposing to HEK cells for one hour, and then diluting the culture medium to a final density of 4 million cells per mL. The cells are then cultured for 7 days with fresh feed medium every 48 hours. After 7 days, the supernatant is collected after centrifugation and purified using protein A. If necessary, CHT column purification is added to reach> 95% monomer.

[0352] Cell binding assay. CD3+ human Jurkat cells (ATCC) and CHO-S cells (Invitrogen / ThermoFisher) were thawed and washed with cold PBSF buffer pH 7.4 (PBS+0.1% BSA, pH 7.4). Approximately 200,000 cells were aliquoted into each well of a 96-well plate (FACS assay plate, VWR BD 353263) and precipitated by centrifugation (5 minutes at 500x g). The cells were washed with PBSF pH 7.4 or PBSF pH 6.0 (PBS+0.1% BSA, pH 6.0), and then resuspended in 100ul of PBSF pH 7.4 or PBSF pH 6.0 with IgG antibodies (100nM) produced in yeast as described above. The mixture (cells+antibody) was incubated on ice for 20 minutes, then washed twice with PBSF pH 7.4 or PBSF pH 6.0. Cells were resuspended in 50ul of propidium iodide (Roche; 1:500 dilution) and anti-human IgG-RPE (1:100 dilution) prepared in PBSF pH 7.4 or PBSF pH 6.0 and then incubated on ice for 20 minutes in the dark, after which the cells were washed twice with PBSF pH 7.4 or PBSF pH 6.0. Binding was analyzed on a FACS Canto II.

[0353] ForteBio KD measurement (biolayer interference measurement; BLI). ForteBio affinity measurements were performed as described previously (Estep, P., et al., High throughput solution-based measurement of antibody-antigen affinity and epitope binning. MAbs, 2013. 5 (2): pp. 270-8.). In brief, ForteBio affinity measurements were performed by loading IgG onto the AHC sensor online. The sensor was balanced offline in the assay buffer for 30 minutes, and then monitored online for 60 seconds to establish a baseline. The sensor with loaded IgG was exposed to 100 nM antigen (e.g., CD3) for 5 minutes, and then they were transferred to the assay buffer for 5 minutes for off-rate measurement. Kinetics were analyzed using a 1:1 binding model.

[0354] PSR preparation. As described in, for example, WO 2014 / 179363 and Xu et al., Protein Eng Des Sel, 26(10):663-670(2013), polyspecific reactive reagents (PSR) are prepared. In brief, 2.5 liters of CHO-S cells are used as starting material. Cells are precipitated at 2,400xg for 5 minutes in a 500mL centrifuge bottle filled to 400mL. The cell pellets are combined and then resuspended in 25ml buffer B and precipitated at 2,400xg for 3 minutes. The buffer is decanted and the washing is repeated once. The cell pellet is resuspended in 3x precipitation volumes of buffer B containing 1x protease inhibitors (Roche, complete, EDTA-free) using a polytron homogenizer, and the cells are kept on ice. 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 that unbroken cells, cell debris and nuclei were removed; the resulting supernatant was a total protein preparation. The supernatant was then transferred to two Nalgene Oak Ridge 45 mL centrifuge tubes and precipitated at 40,000 x g for 40 minutes at 4 ° C. The supernatant containing the isolated cytosolic protein (SCP) was then transferred to a clean Oak Ridge tube and centrifuged once more at 40,000 x g. In parallel, the precipitate containing the membrane fraction (EMF) was retained and centrifuged at 40,000 for 20 minutes to remove the residual supernatant. The EMF precipitate was then rinsed with buffer B. 8 mL of buffer B was then added to the membrane precipitate to remove the precipitate and transferred to a Dounce homogenizer. After homogenizing the precipitate, they were transferred to a 50 mL conical tube and represented the final EMF preparation.

[0355] Will be about 10 6 –10 71 billion mammalian cells (e.g., CHO, HEK293, Sf9) of each cell / mL are transferred to 4x 250mL conical tubes from a tissue culture environment and precipitated at 550x g for 3 minutes. All subsequent steps are performed at 4°C or on ice with ice-cold buffer. The cells are washed with 100mL PBSF (1x PBS+1mg / mL BSA) and merged into a conical tube. After removing the supernatant, the cell pellet is then resuspended in 30mL buffer B (50mM HEPES, 0.15MNaCl, 2mM CaCl2, 5mM KCl, 5mM MgCl2, 10% glycerol, pH 7.2) and precipitated at 550x g for 3 minutes. The buffer B supernatant is decanted, and the cells are resuspended in the buffer B of 3x precipitation volume plus 2.5x protease inhibitors (Roche, completely, EDTA-free). The protease inhibitors in buffer B are included from here onward. The cells were homogenized four times for 30 second pulses (Polyton 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 represented by 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 moved to a new collection tube and the pestle was rinsed to collect all potential proteins. The protein concentration of the purified EMF was determined using the Dc-Protein Assay Kit (BioRad). To solubilize EMF, transfer to a lysis buffer (50 mM HEPES, 0.15 M NaCl, 2 mM CaCl2, 5 mM KCl, 5 mM MgCl2, 1% n-dodecyl-bD-maltosylpyranoside (DDM), 1x protease inhibitor (pH 7.2) to a final concentration of 1 mg / mL. The mixture was rotated overnight at 4°C with rotation, followed by centrifugation at 40,000 x g for 1 hour in a 50 mL OakRidge tube (Fisher Scientific, 050529-ID). The supernatant representing soluble membrane protein (SMP) was collected and the protein yield was quantified as described above.

[0356] For biotinylation, NHS-LC-biotin stock solution was prepared according to the manufacturer's scheme (Pierce, Thermo Fisher). In brief, 20ul biotin reagent was added to every 1mg EMF sample, and incubated for 3 hours at 4°C with gentle stirring. The volume was adjusted to 25mL with buffer B, and transferred to an Oak Ridge centrifuge tube. Biotinylated EMF (b-EMF) was precipitated for 1 hour at 40,000xg, and rinsed twice with 3mL buffer C (buffer B minus glycerol) without interfering with the precipitation. Residual solution was removed. As previously described, the precipitation was resuspended in 3mL buffer C with a Dounce homogenizer. The resuspended precipitation now represents biotinylated EMF (b-EMF). Dissolve to prepare b-SMP as described above.

[0357] PSR binding analysis. Generally, the assay is performed as described in, for example, the following literature: Xu et al. Protein Eng Des Sel, 26 (10): 663-670 (2013). In order to characterize the PSR spectrum of monoclonal antibodies presented on yeast, 2 million yeasts presenting IgG were transferred to a 96-well assay plate and precipitated at 3000 x g for 3 minutes to remove the supernatant. The precipitate was resuspended in 50ul of a freshly prepared 1:10 diluted b-PSR stock solution and incubated on ice for 20 minutes. The cells were washed twice with 200ul cold PBSF, and the precipitate was resuspended in 50ul secondary labeling mixture (Extravidin-R-PE, anti-human LC-FITC and propidium iodide). The mixture was incubated on ice for 20 minutes, followed by washing twice with 200ul ice-cold PBSF. The cells were resuspended in 100ul ice-cold PBSF, and the plate was run on FACSCanto (BD Biosciences) using an HTS sample injector. Flow cytometry data were analyzed for mean fluorescence intensity in the R-PE channel and normalized to appropriate controls to assess nonspecific binding. Many methods for presenting or displaying antibodies or antibody fragments on the surface of yeast have been described previously, all of which are consistent with the present protocol (Blaise et al., Gene, 342(2):211-8 (2004), Boder and Wittrup, Nat Biotechnol., 15(6):553-7 (1997), Kuroda and Ueda, Biotechnol Lett., 33(1):1-9 (2011), Orcutt and Wittrup, Springer Protocols: Antibody Engineering, 1:207-233 (2010), Rakestraw et al., Protein Eng Des Sel., 24(6):525-30 (2011), Sazinsky et al., Proc Natl Acad Sci US A., 105(51):20167-72 (2008), Tasumi et al., Proc Natl Acad Sci US A., 105(51):20167-72 (2008), A., 106(31):12891-6(2009)).

[0358] ForteBio Kinetics. The FortBio Octet HTX instrument is used in 12-channel mode (8 sensors per channel, 96 sensors per experiment) equipped with AHC, SA or AHQ sensors. The instrument is driven by software (versions 8.2 and 9.0) supplied by the manufacturer. The sample name and concentration are entered into the plate data page, and the sensor-associated protein is identified in the "Information" column on the sensor data page. Kinetic experiments are collected with a 90 or 180 s baseline, a 180 s association phase, and a 180 s dissociation phase. All files are saved to a shared network drive with a naming convention that identifies the experimental format.

[0359] The IgG1 sample buffer was exchanged into 1M ammonium sulfate and 0.1M sodium phosphate (pH 6.5) using Zeba 40kDa 0.5mL spin column (Thermo Pierce, catalog number 87766). A salt gradient from 1.8M ammonium sulfate, 0.1M sodium phosphate (pH 6.5) to the same conditions without ammonium sulfate was established on a Dionex ProPac HIC-10 column. The gradient was run for 17 minutes at a flow rate of 0.75ml / minute. An acetonitrile wash step was added at the end of the run to remove any remaining protein, and the column was rebalanced in 7 column volumes before the next injection cycle. Peak retention time was monitored under A280 absorbance, and ammonium sulfate concentration during elution was calculated based on gradient and flow rate.

[0360] LCMS. mAb samples were reduced by DTT and subsequently analyzed by mid-drop LCMS on a BrukermaXis4G mass spectrometer coupled to an Agilent 1100HPLC (Agilent). A POROS R2 10 μm (2.1 x 30 mm) reverse phase column was used to remove salts from the sample. The fast LC flow rate at 2 mL / min allowed separation of the sample and salts, elution of the sample, and regeneration of the column to be completed within a 2.1 minute cycle. A T-junction was used to deliver only 0.15 mL / min of sample flow to the mass spectrometer for sample analysis. The BrukermaXis 4G mass spectrometer was operated in positive ion mode and detected in the range of 750 to 2500 m / z. The remaining source parameters were set as follows; the capillary was set to 5500 V, the nebulizer was set to 4.0 bar, the drying gas was set to 4.0 l / min, and the drying temperature was set to 200C.

[0361] MS spectra were analyzed using Bruker Data Analysis version 4.1, and deconvolution was done using maximum entropy deconvolution in the mass range of 20 to 30 kDa.

[0362] An informal sequence listing is provided in Table 1. The informal sequence listing provides the heavy chain variable region ("HC") amino acid sequence, in which each of the heavy chain variable region CDRs is underlined, and the light chain variable region ("LC"), in which each of the light chain variable region CDRs is underlined.

[0363]

[0364]

[0365]

[0366]

[0367]

[0368]

[0369]

[0370]

[0371]

[0372]

[0373]

[0374]

[0375]

[0376]

[0377]

[0378]

[0379]

[0380]

[0381]

[0382]

[0383]

[0384]

[0385]

[0386]

[0387]

[0388]

[0389]

[0390]

[0391]

[0392]

[0393]

[0394]

[0395]

[0396]

[0397]

[0398]

[0399]

[0400]

[0401]

[0402]

[0403]

[0404]

[0405]

[0406]

[0407]

[0408]

[0409]

[0410]

[0411]

[0412]

[0413]

[0414]

[0415]

[0416]

[0417]

[0418]

[0419]

[0420]

[0421]

[0422]

[0423]

[0424]

[0425]

[0426] Table 4. Human and cynomolgus monkey CD3ε sequences

[0427]

Claims

1. An anti-cluster of differentiation 3 ("CD3") antibody and / or antigen-binding fragment, the anti-CD3 antibody and / or antigen-binding fragment comprising: (a) comprising V selected from the following H The variable heavy (V H ) chain polypeptide: i.AX1DX2X3X4X5X6X7X8DX9, wherein X1 is R or H, wherein X2 is A, H, M or Q, wherein X3 is Y, H, S, G, A, T, V or R; wherein X4 is G, H, P, E or R; wherein X5 is H or R, wherein X6 is Y, N, F, H, D, E, S, L, M, I, G, A, Q or T; wherein X7 is F or H; wherein X8 is Y or H; wherein X9 is V, H or M; and optionally wherein at least one of X1, X2, X3, X4, X5, X6, X7, X8 and X9 is H (SEQ ID NO: 58); ii.ARDX1X2X3X4YFYDX5, wherein X1 is H or A, wherein X2 is T, Y or H, wherein X3 is G or H, wherein X4 is H, R, V or I, wherein X5 is V or H, and optionally wherein at least one of X1, X2, X3, X4 and X5 is H (SEQ ID NO: 43); iii.AX1DX2YX3HX4FYDV, wherein X1 is R or H, wherein X2 is A or H, wherein X3 is G, H or P, wherein X4 is Y, H, D, V, E, S, N, L, M, I, G, A, Q or T, and optionally wherein at least one of X1, X2, X3 and X4 is H (SEQ ID NO: 1); iv.ARDX1YGX2X3X4YDX5, wherein X1 is A or H, wherein X2 is R or H, wherein X3 is H or Y, wherein X4 is F or H, wherein X5 is H or V, and optionally wherein at least one of X1, X2, X3, X4 and X5 is H (SEQ ID NO: 2): v.ARDAHX1X2YX3X4DX5, wherein X1 is G, E or R, wherein X2 is R or H, wherein X3 is F or H, wherein X4 is Y or H, wherein X5 is V or H, and optionally wherein at least one of X1, X2, X3, X4 and X5 is H (SEQ ID NO: 3); vi.ARDAX1HRX2FYDV, wherein X1 is H, Y, S, G, A, T, V or R, wherein X2 is Y or H, and optionally wherein at least one of X1 and X2 is H (SEQ ID NO: 4); vii.ARDX1YHRYFYDX2, wherein X1 is H or A, wherein X2 is H, V or M, and optionally wherein at least one of X1 and X2 is H (SEQ ID NO: 5); viii.AX1DAYX2X3X4HX5DV, wherein X1 is R or H, wherein X2 is G or H, wherein X3 is H or R, wherein X4 is N, F or Y, wherein X5 is Y or H, and optionally wherein at least one of X1, X2, X3, X4 and X5 is H (SEQ ID NO: 6); ix.ARDX1X2GRYFYDV, wherein X1 is M, Q or H, wherein X2 is R or H, and optionally wherein at least one of X1 and X2 is H (SEQ ID NO: 7); x.ARDX1X2X3RYFYDX4, wherein X1 is H or A, wherein X2 is T, Y or H, wherein X3 is G or H, wherein X4 is V or H, and optionally wherein at least one of X1, X2, X3 and X4 is H (SEQ ID NO: 8); xi.ARDAX1X2X3X4FYDX5, wherein X1 is T, H or Y, wherein X2 is G or H, wherein X3 is H or R, wherein X4 is V or Y, wherein X5 is V or H, and wherein optionally at least one of X1, X2, X3 and X5 is H (SEQ ID NO: 593); and xii.AX1DX2X3X4X5X6X7YDX8, wherein X1 is R or H, wherein X2 is H or A, wherein X3 is H or Y, wherein X4 is H, G or P, wherein X5 is R or H, wherein X6 is Y, I or V, wherein X7 is F or H, wherein X8 is V or H, and wherein optionally at least one of X1, X2, X3, X4, X5, X7 and X8 is H (SEQ ID NO: 596). (b) comprising V selected from the following H The variable heavy (V H ) chain polypeptide: i. X1IX2X3X4X5X6X7TX8YSQKFQG, wherein X1 is W, S, Y, F, G or D, wherein X2 is N, T, D, V or H, wherein X3 is A, P or S, wherein X4 is G, A, S, N, D, L, V, H, Q, T, I or Y, wherein X5 is D or T, wherein X6 is A or G, wherein X7 is A, N, T, S, L, D, F, Y or E, wherein X8 is V, K, T, D, Y, F, A, H, N, L, I or E, and optionally wherein at least one of X1, X2, X3, X4, X5, X6, X7 and X8 is H (SEQ ID NO: 59); ii.WIDLENANTIYDAKFQG(SEQ ID NO:9); iii. X1INPX2TGX3TX4YSQKFQG, wherein X1 is W or Y, wherein X2 is A, S, D, G, N, L, V, H or Q, wherein X3 is A, T or S, and wherein X4 is K, V, T, D, Y, F or A (SEQ ID NO: 10); iv. X1IX2AGTGX3TX4YSQKFQG, wherein X1 is W, Y or F, wherein X2 is T, N or D, wherein X3 is A, T or L, and wherein X4 is A, K, V, H, T or N (SEQ ID NO: 11); v. X1IDAGTGX2TX3YSQKFQG, wherein X1 is S or W, wherein X2 is L, N, D or F, and wherein X3 is D, Y or K (SEQ ID NO: 12); vi.X1IX2AGTGATX3YSQKFQG, wherein X1 is G, D or S, wherein X2 is I or D, and wherein X3 is K or D (SEQ ID NO: 13); vii. WINPX1TGNTX2YSQKFQG, wherein X1 is D, T, L, S or A, and wherein X2 is D, V, L or N (SEQ ID NO: 14); viii. X1INAGTGX2TX3YSQKFQG, wherein X1 is Y or W, wherein X2 is N, D or A, and wherein X3 is I or V (SEQ ID NO: 15); ix. X1INPX2TGX3TKYSQKFQG, wherein X1 is W or Y, wherein X2 is D, I or Y, and wherein X3 is D, Y or E (SEQ ID NO: 16); x.SIX1AGTGX2TKYSQKFQG, wherein X1 is N or V, and wherein X2 is A or I (SEQ ID NO: 17); xi.SINAGTGX1TX2YSQKFQG, wherein X1 is F or N, and wherein X2 is Y or D (SEQ ID NO: 18); xii.X1IX2X3GTGX4TDYSQKFQG, wherein X1 is D or W, wherein X2 is N or H, wherein X3 is A or S, and wherein X4 is A or N (SEQ ID NO: 19); xiii.WIDPX1TGATX2YSQKFQG, wherein X1 is N, H or Y, and wherein X2 is V or K (SEQ ID NO: 20); xiv.WIX1PX2TGNTKYSQKFQG, wherein X1 is D or N, and wherein X2 is L, I or V (SEQ ID NO: 21); xv.SINAGDANTKYSQKFQG(SEQ ID NO:22); xvi.X1IDPX2TGATX3YSQKFQG, wherein X1 is D or W, wherein X2 is D or V, and wherein X3 is E or D (SEQ ID NO: 23); xvii. WINAGDAATVYSQKFQG (SEQ ID NO: 24); and xviii. WIDAGTGX1TX2YSQKFQG, wherein X1 is L, F, N or A, and wherein X2 is T or K (SEQ ID NO: 595). (c) comprising V selected from the following H The variable heavy (V H ) chain polypeptide: i. FNIKDYHMH (SEQ ID NO: 25); ii. SNIKDYYMH (SEQ ID NO: 26); iii.SNIKDYHMH (SEQ ID NO:27); iv. YTFX1X2X3X4MH, wherein X1 is A, K, D, Q, E, N, T, L, Y, S, P, G, H or V, wherein X2 is T, S or A, wherein X3 is Y or I, and wherein X4 is A, D, N, S, Y, T, I, V, L, E, P, R or G (SEQ ID NO: 28); v.YTFX1X2X3X4MH, wherein X1 is T, D, A, N or V, wherein X2 is D, E, G or Q, wherein X3 is Y or D, and wherein X4 is D, A, E, N, S, Y or V (SEQ ID NO: 29); vi. YTFTSX1X2MH, wherein X1 is A, D or T, and wherein X2 is D, F, A, M, V or Y (SEQ ID NO: 30); vii. YTFX1X2YX3MH, wherein X1 is N or T, X2 is Q or N, and X3 is S, T or A (SEQ ID NO: 31); and viii. YTFX1X2YVMH, wherein X1 is I or N, and wherein X2 is K or R (SEQ ID NO: 32); ix. FNIKDYYMH (SEQ ID NO: 47); and x.YTFX1X2YX3MH, wherein X1 is E, S or T, wherein X2 is S or D, and wherein X3 is A or D (SEQ ID NO: 31). (d) comprising V selected from the following L The variable light (V L ) chain polypeptide: i. X1X2SX3X4X5RX6, wherein X1 is H, K or G, wherein X2 is Q or H, wherein X3 is Y or H, wherein X4 is S, H, D, T, V, M or L, wherein X5 is R or H, wherein X6 is T or H, and optionally wherein at least one of X1, X2, X3, X4, X5 and X6 is H (SEQ ID NO: 33); ii. KQSYX1X2RT, wherein X1 is H, V, K, W, R, L, G, Y or Q, wherein X2 is H, L, E, W, G, M, P, T, Q or V, and optionally at least one of X1 and X2H (SEQ ID NO: 34); iii. X1QSX2HX3RT, wherein X1 is K or H, wherein X2 is H, Y, M, S, L, E, G or W, wherein X3 is R or K, and optionally wherein at least one of X1 and X2 is H (SEQ ID NO: 35); iv. KQSX1X2X3RT, wherein X1 is Y or H, wherein X2 is T, S, V or K, wherein X3 is R or H, and optionally wherein at least one of X1 and X3 is H (SEQ ID NO: 36); v. KQSX1X2X3RT, wherein X1 is H or Y, wherein X2 is T, S or Q, wherein X3 is R or H, and optionally wherein at least one of X1 and X3 is H (SEQ ID NO: 36); and vi.X1QSX2X3X4RT, wherein X1 is K or H, wherein X2 is Y or H, wherein X3 is S, H, L, V or K, wherein X4 is H, R or E, and optionally wherein at least one of X1, X2, X3 and X4 is H (SEQ ID NO: 598); (e) V comprising WASTRES (SEQ ID NO: 37) L The variable light (V L ) chain polypeptide; and / or (f) comprising V selected from the following L Variable light (V L ) chain polypeptide: i.KSSQSLLX1X2X3X4GX5NX6LA, wherein X1 is N or H, wherein X2 is A, R or T, wherein X3 is R or H, wherein X4 is T, P or E, wherein X5 is H or K, wherein X6 is H or Y, and optionally wherein at least one of X1, X3, X5 and X6 is H (SEQ ID NO: 38); ii. KSSQSLLX1AX2THX3NX4LA, wherein X1 is N or H, wherein X2 is R or H, wherein X3 is K or H, wherein X4 is Y or H, and optionally wherein at least one of X1, X2, X3 and X4 is H (SEQ ID NO: 39); iii.KSSQSLLNASTAKNYLA(SEQ ID NO:40); iv.KSSQSLLNARTRTNYLA(SEQ ID NO:41); v.KSSQSLLNX1X2X3GX4NX5LA, wherein X1 is S or A, wherein X2 is R or H, wherein X3 is E or T, wherein X4 is H or K, wherein X5 is H or Y, and optionally wherein at least one of X2, X4 and X5 is H (SEQ ID NO: 42); vi.KSSQSLLNX1X2TGX3NYLA, wherein X1 is A or S, wherein X2 is R or H, wherein X3 is H or K, and optionally wherein at least one of X2 and X3 is H (SEQ ID NO: 594); and vii.KSSQSLLX1AX2X3X4X5NX6LA, wherein X1 is N or H, wherein X2 is R or H, wherein X3 is T or E, wherein X4 is G or H, wherein X5 is H or K, wherein X6 is H or Y, and wherein optionally at least one of X1, X2, X4, X5 and X6 is H (SEQ ID NO: 597).

2. The anti-CD3 antibody and / or antigen-binding fragment according to claim 1, comprising: (a) a CDRH3 comprising the amino acid sequence ARDX1X2X3X4YFYDX5, wherein X1 is H or A, wherein X2 is T, Y or H, wherein X3 is G or H, X4 is H, R, V or I, wherein X5 is V or H, and optionally wherein at least one of X1, X2, X3, X4 and X5 is H (SEQ ID NO: 43); (b) CDRL3 comprising the amino acid sequence KQSX1X2X3RT, wherein X1 is Y or H, X2 is T, S, V or K, X3 is R or H, and optionally wherein at least one of X1 and X3 is H (SEQ ID NO: 36); and / or (c) CDRL1 comprising the amino acid sequence KSSQSLLNX1X2X3GX4NX5LA, wherein X1 is S or A, wherein X2 is R or H, wherein X3 is E or T, wherein X4 is H or K, wherein X5 is H or Y, and optionally wherein at least one of X2, X4 and X5 is H (SEQ ID NO: 42).

3. The anti-CD3 antibody or antigen-binding fragment according to claim 1, comprising: (a) a CDRH3 comprising the amino acid sequence ARDAX1XXXFYDX5, wherein X1 is T, H or Y, wherein X2 is G or H, wherein X3 is H or R, wherein X4 is V or Y, wherein X5 is V or H, and wherein optionally at least one of X1, X2, X3 and X5 is H (SEQ ID NO: 593); (b) a CDRH2 comprising the amino acid sequence WIDLENANTIYDAKFQG (SEQ ID NO: 9); (c) a CDRH1 comprising the amino acid sequence FNIKDYYMH (SEQ ID NO: 47); (d) CDRL3 comprising the amino acid sequence KQSX1X2X3RT, wherein X1 is H or Y, wherein X2 is T, S or Q, wherein X3 is R or H, and optionally wherein at least one of X1 and X3 is H (SEQ ID NO: 36); (e) CDRL2 comprising the amino acid sequence WASTRES (SEQ ID NO: 37); and / or (f) CDRL1 comprising the amino acid sequence KSSQSLLNX1X2TGX3NYLA, wherein X1 is A or S, wherein X2 is R or H, wherein X3 is H or K, and optionally wherein at least one of X2 and X3 is H (SEQ ID NO: 594).

4. The anti-CD3 antibody or antigen-binding fragment according to claim 1, comprising: (a) a CDRH3 comprising the amino acid sequence AX1DX2XXXX5X6X7YDX8, wherein X1 is R or H, wherein X2 is H or A, wherein X3 is H or Y, wherein X4 is H, G or P, wherein X5 is R or H, wherein X6 is Y, I or V, wherein X7 is F or H, wherein X8 is V or H, and wherein optionally at least one of X1, X2, X3, X4, X5, X7 and X8 is H (SEQ ID NO: 596); (b) a CDRH2 comprising an amino acid sequence selected from the group consisting of: i.WIDLENANTIYDAKFQG (SEQ ID NO:9); and ii. WIDAGTGX1TX2YSQKFQG, wherein X1 is L, F, N or A, and wherein X2 is T or K (SEQ ID NO: 595); (c) a CDRH1 comprising an amino acid sequence selected from the group consisting of: i.FNIKDYYMH (SEQ ID NO:47); and ii. YTFX1X2YX3MH, wherein X1 is E, S or T, wherein X2 is S or D, and wherein X3 is A or D (SEQ ID NO: 31); (d) CDRL3 comprising the amino acid sequence X1QSX2XXXRT, wherein X1 is K or H, wherein X2 is Y or H, wherein X3 is S, H, L, V or K, wherein X4 is H, R or E, and optionally wherein at least one of X1, X2, X3 and X4 is H (SEQ ID NO: 598); (e) CDRL2 comprising the amino acid sequence WASTRES (SEQ ID NO: 37); and / or (f) CDRL1 comprising the amino acid sequence KSSQSLLX1AX2X3X4X5NX6LA, wherein X1 is N or H, wherein X2 is R or H, wherein X3 is T or E, wherein X4 is G or H, wherein X5 is H or K, wherein X6 is H or Y, and wherein optionally at least one of X1, X2, X4, X5 and X6 is H (SEQ ID NO: 597).

5. The anti-CD3 antibody or antigen-binding fragment according to any one of the preceding claims, wherein: (a) the antibody or antigen-binding fragment induces T cell activation or T cell killing, while showing a reduced tendency to induce cytokine production to a level capable of inducing cytokine release syndrome; (b) the antibody or antigen-binding fragment comprises a multispecific antibody; (c) the antibody or antigen-binding fragment comprises a bispecific antibody; (d) the antibody or antigen-binding fragment comprises a scFv; (e) the antibody or antigen-binding fragment comprises at least a second antigen-binding domain that specifically binds to an oncology 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; (f) the antibody or antigen-binding fragment comprises at least a second antigen-binding domain that specifically binds to an antigen selected from the group consisting of: 17-IA, 4-1BB, 4Dc, 6-keto-PGF1a, 8-iso-PGF2a, 8-oxo-dG, Al adenosine receptor, A33, ACE, ACE-2, activin, activin A, activin AB, activin B, activin C, activin RIA, activin RIA ALK-2, activin RIB ALK-4, activin RIIA, activin RUB, ADAM, ADAM10, ADAM12, ADAM 15, ADAM 17 / TACE, ADAM8, ADAM9, ADAMTS, ADAMTS4, ADAMTS5, addressin, aFGF, ALCAM, ALK, ALK-1, ALK-7, alpha-l-antitrypsin, alpha-V / beta-1 antagonist, ANG, Ang, APAF-1, APE, APJ, APP, APRIL, AR, ARC, ART, artesunate, anti-Id, ASPARTIC, atrial natriuretic factor, av / b3 integrin, Ax l, 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-2BMP-2a, BMP-3osteomorphin, BMP-4BMP- 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), cancer-associated antigen, cathepsin A, cathepsin B, cathepsin C / DPPI, cathepsin D, cathepsin E, cathepsin H, cathepsin L, cathepsin O, cathepsin S, cathepsin V, cathepsin X / Z / P, CBL, CCI, CCK2, CCL, CCL1, CCL11, CCL12, CCL13, CCL 14, CCL15, CCL16, CCL17. CCL18, CCL19, CCL2, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9 / 10, CCR, CCR1, CCR10, CCR10, CC R2, 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, C D23, 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, Clostridium botulinum toxin, Clostridium perfringens 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, CXC L4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCR, CXCR1, CX CR2, CXCR3, CXCR4, CXCR5, CXCR6, cytokeratin tumor-associated antigen, DAN, DCC, DcR3, DC-SIGN, decay-accelerating factor, des(l-3)-IGF-I (brain IGF-1), Dhh, digoxin, DNAM-1, Dnase, Dpp, DPPIV / CD26, Dtk, ECAD, EDA, EDA-A1, EDA-A2, EDAR, EGF, EGFR (ErbB-1), EMA, EMMPRIN, ENA, endothelin receptor, enkephalinase, eNOS, Eot, eotaxin 1, 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, fibrinogen, 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-α1, GFR-α2, GFR-α3, 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), 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, HIVIIIB gp120V3 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-18 R, IL-23, interferon (INF)-α, INF-β, INF-γ, inhibin, iNOS, insulin A chain, insulin B chain, insulin-like growth factor 1, integrin α2, integrin α3, integrin α4, integrin α4 / βl, integrin, α4 / β7, integrin α5 (αV), integrin α5 / βl, integrin α5 / β3, integrin α6, integrin βl, integrin β2, interferon γ, IP-10, 1-TAC, JE, kallikrein 2, kallikrein 5, kallikrein 6, kallikrein 11, kallikrein 12, kallikrein 14, kallikrein 15, kallikrein LI, kallikrein L2, kallikrein Kallikrein L3, Kallikrein L4, KC, KDR, Keratinocyte Growth Factor (KGF), Laminin 5, LAMP, LAP, LAP (TGF-1), Potential TGF-1, Potential TGF-1bpl, LBP, LDGF, LECT2, Lefty, Lewis-Y Antigen, Lewis-Y Related Antigen, LFA-1, LFA-3, Lfo, LIF, LIGHT, Lipoprotein, LIX, LKN, Lptn, L-Selectin, LT-a, LT-b, LTB4, LTBP-1, Pulmonary Surfactant, Luteinizing Hormone, Lymphotoxin B Receptor, Mac-1, MAdCAM, MAG, MAP2, MARC, MCAM, MCAM, MCK-2, MCP, M-CSF, MDC, Mer, α-metalloproteinase, MGDF receptor, MGMT, MHC (HLA-DR), MIF, MIG, MIP, MIP-1-α, 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, Mullerian-inhibiting substance (MUC18),substance), Mug, MuSK, NAIP, NAP, NCAD, N-cadherin, NCA 90, NCAM, NCAM, Neprilysin, neurotrophin-3, -4 or -6, neurotrophin (Neurturin), neuron growth factor (NGF), NGFR, NGF-β, nNOS, NO, NOS, Npn, NRG-3, NT, NTN, OB, OGG1, OPG, OPN, OSM, OX40L, OX40R, pl50, p95, PADPr, parathyroid hormone, PARC, PARP, PBR, PBSF, PCAD, P-cadherin, PCNA, PDGF, PDGF, PDK-1, PECAM, PEM, PF4, PGE, PGF, PGI2, PGJ2, PIN, PLA2, placental alkaline phosphatase (PLAP), PIGF, PLP, PP14, proinsulin, prorelaxin, protein C, PS, PSA, PSCA, prostate-specific membrane antigen (PSMA), PTEN, PTHrp, Ptk, PTN, R51, RANK, RANKL, RANTES, relaxin A chain, relaxin B chain, renin, respiratory syncytial virus (RSV) F, 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 receptors (e.g., T cell receptor α / β), TdT, TECK, TEM1, TEM5, TEM7, TEM8, TERT, testis P LAP-like alkaline phosphatase, TfR, TGF, TGF-α, TGF-β, TGF-βPan specific, TGF-βRI (ALK-5), TGF-βRII, TGF-βRllb, TGF-βRIII, TGF-βl, TGF-β2, TGF-β3, TGF-β4, TGF-β5, thrombin, thymus Ck-1, thyroid stimulating hormone, Tie, TIMP, TIQ, tissue factor, TMEFF2, Tmpo, TMPRSS2, TNF, TNF-α, TNF-αβ, TNF-β2, TNFc, TNF-RI, TNF-RII, TNFRSF10A (TRAIL Rl Apo-2, DR4), TNFRSFIOB (TRAIL R2 DR5, KILLER, TRICK-2A, TRICK-B), TNFRSF10C (TRAIL R3DcRl、LIT、TRID)、TNFRSF10D(TRAIL R4DcR2、TRUNDD)、TNFRSF11A(RANK ODF R、TRANCE R)、TNFRSFllB(OPG OCIF、TR1)、TNFRSF12(TWEAK R). FN14)、TNFRSF13B(TACI)、TNFRSF13C(BAFF R)、TNFRSF14(WHEM ATAR、HveA、LIGHT R、TR2)、TNFRSF16(NGFR p75NTR)、TNFRSF17(BCMA)、TNFRSF 18(GITR). AITR)、TNFRSF19(TROY CROWN、TRADE)、TNFRSF19L(RELT)、TNFRSFIA(TNF RI CD120a、p55-60)、TNFRSFIB(TNF RIICD120b, p75-80, TNFRSF26(TNFRH3), TNFRSF3(LTbR TNF RIII, TNFCR), TNFRSF4(OX40 ACT35, TXGP1 R), TNFRSF 5(CD40 p50), TNFRSF6(Fas). Apo-1, APT1, CD95, TNFRSF6B(DcR3 M68, TR6), TNFRSF7(CD27), TNFRSF8(CD30), TNFRSF9(4-1BB CD137, ILA), TNFRSF21(DR6), TNFRSF22(DcTRAIL R2). TNFRH2, TNFRST23(DcTRAIL Rl TNFRH1), TNFRSF25(DR3 Apo-3, LARD, TR-3, TRAMP, WSL-1), TNFSF10(TRAIL Apo-2 division, TL2, TNFSF11 (TRANCE / RANK division ODF, OPG division), TNFSF12 (TWEAK Apo-3 division, DR3 division), TNFSF13 (APRIL TALL2), TNFSF13B (BAFF). LIGHT、TALL1、THANK、TNFSF20)、TNFSF14(LIGHT HVEM trigger, LTg, TNFSF15(TLIA / VEGI), TNFSF18 (GITR trigger AITR trigger, TL6), TNFSFIA (TNF-dependent enzyme (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, transfer receptor, TRF, Trk, TROP-2, TSG, TSLP, tumor-associated antigen CA 125, tumor-associated antigen expressing Lewis Y-related carbohydrate carbohydrate), 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, anti-von Willebrand factor (von Willebrands 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 3), hormone receptors and growth factors; (g) the antibody or antigen-binding fragment 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 receptor (TLR), TLR4, TLR9, cytokines, IL-2, IL-5, IL-13, IL-6, IL-17, IL-12, IL-23, TNFa, TGFb, cytokine receptors, IL-2R, chemokines, chemokine receptors, growth factors, VEGF, and HGF; (h) the antibody or antigen-binding fragment is comprised in a chimeric antigen receptor (CAR), which optionally comprises at least one transmembrane domain, and at least one intracellular domain from a T cell receptor, optionally the CD3 zeta subunit, and at least one co-stimulatory domain; (i) the antibody or antigen-binding fragment comprises scFv2-Fc2 and / or scFv-IgG; (j) the antibody or antigen-binding fragment comprises an IgG constant domain; and / or (k) the antibody or antigen-binding fragment comprises at least a second antigen-binding domain that specifically binds to an antigen, wherein the antibody comprises a multispecific format selected from the group consisting of Fab-Fc-scFv, "bottle-opener", Mab-scFv, Mab-Fv, dual scFv, central Fv, central scFv, one-armed central scFv, Fab-Fab, Fab-Fv, mAb-Fv, mAb-Fab, DART, BiTE, common light chain-IgG, TandAb, Cross-Mab, SEED, BEAT, TrioMab and DuetMab.

6. The anti-CD3 antibody and / or antigen-binding fragment of any one of the preceding claims, which binds to CD3 or a cell expressing CD3 with greater binding affinity at pH 6.0 than at pH 7.

4.

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

8. An expression vector comprising the isolated or recombinant nucleic acid sequence according to claim 7.

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

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

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

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

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