ANTI-CD3 ANTIBODIES AND USES THEREOF

SEQUENCE LISTING

The present application hereby incorporates by reference the entire contents of the XML file named “206389-0062-01US SequenceListing.xml” in XML format, which was created on Mar. 2, 2022, and is 638,488 bytes in size.

TECHNICAL FIELD

The disclosure provided herein relates to anti-cluster of differentiation 3 (CD3)-antibodies, and antigen-binding fragments thereof, capable of specifically binding to human and non-human CD3, and in particular to anti-CD3 antibodies and antigen-binding fragments that are cross-reactive with CD3 of a non-human mammal (e.g., a cynomolgus monkey); prostate specific membrane antigen (PSMA)-antibodies, and antigen-binding fragments thereof, capable of specifically binding to human and non-human PSMA; IL1RAP antibodies, and antigen-binding fragments thereof, capable of specifically binding to human and non-human IL1RAP; CD33 antibodies, and antigen-binding fragments thereof, capable of specifically binding to human and non-human CD33; and bispecific antibodies that are capable of specifically binding CD3; PSMA; IL1RAP; CD33; CD3 and PSMA; CD3 and IL1RAP; or CD3 and CD33. The disclosure also pertains to uses of such antibodies and antigen-binding fragments in the treatment of cancer, autoimmune and/or inflammatory diseases and other conditions.

BACKGROUND

Bispecific antibodies and antibody fragments have been explored as a means to recruit cytolytic T cells to kill tumor cells. However, the clinical use of many T cell-recruiting bispecific antibodies has been limited by challenges including unfavorable pharmacokinetics, potential immunogenicity, and manufacturing issues. There thus exists a considerable need for bispecific antibodies that recruit cytolytic T cells to kill tumor cells that exhibit reduced toxicity and favorable manufacturing profiles.

The human CD3 T cell antigen receptor protein complex is composed of six distinct chains: a CD3γ chain (SwissProt P09693), a CD3δ chain (SwissProt P04234), two CD3ε chains (SwissProt P07766), and one CD3ζ chain homodimer (SwissProt P20963) (εγ:εδ:ζζ), which is associated with the T cell receptor α and δ chain. This complex plays an important role in coupling antigen recognition to several intracellular signal-transduction pathways. The CD3 complex mediates signal transduction, resulting in T cell activation and proliferation. CD3 is required for immune response.

SUMMARY

Provided herein are isolated recombinant anti-CD3 antibodies, or antigen-binding fragments thereof, comprising: a heavy chain comprising a heavy chain complementarity determining region (HCDR) 1 comprising SEQ ID NO: 662; a HCDR2 comprising SEQ ID NO: 663; and a HCDR3 comprising SEQ ID NO: 664 and a light chain comprising a light chain complementarity determining region (LCDR) 1 comprising SEQ ID NO: 671, a LCDR2 comprising SEQ ID NO: 673, and a LCDR3 comprising SEQ ID NO: 690; a heavy chain variable region comprising SEQ ID NO: 652 and a light chain variable region comprising SEQ ID NO: 661; a heavy chain comprising SEQ ID NO: 640 and a light chain comprising SEQ ID NO: 676; or comprising: a heavy chain comprising a HCDR1 comprising SEQ ID NO: 662; a HCDR2 comprising SEQ ID NO: 663; and a HCDR3 comprising SEQ ID NO: 664 and a light chain comprising a LCDR1 comprising SEQ ID NO: 773, a LCDR2 comprising SEQ ID NO: 673, and a LCDR3 comprising SEQ ID NO: 690; a heavy chain variable region comprising SEQ ID NO: 657 and a light chain variable region comprising SEQ ID NO: 678; or a heavy chain comprising SEQ ID NO: 675 and a light chain comprising SEQ ID NO: 678.

Also provided are isolated recombinant anti-CD3 antibodies or antigen-binding fragments thereof, that specifically bind Macaca fascicularis or human CD3d, or CD3e, or CD3e and CD3d with a binding affinity of about 300 nM or less.

In some embodiments, the isolated recombinant anti-CD3 antibodies or antigen-binding fragments thereof have one, two, three, or four, of the following properties:

- bind human and Macaca fascicularis CD3+T lymphocytes with a calculated EC50 of 20 nM or less and bind Macaca fascicularis CD3-expressing HEK cells with a calculated EC50 of 40 nM or less, wherein the difference in calculated EC50 between binding CD3+T lymphocytes and binding Macaca fascicularis CD3-expressing HEK cells is less than 5-fold, and wherein the calculated EC50 is measured in a whole cell binding assay at 0° C. using flow cytometry;
- bind recombinant CD3d from human (SEQ ID NO:691), or bind recombinant CD3e from human (SEQ ID NO:636), or recombinant CD3d from Macaca fascicularis (SEQ ID NO:692), with an equilibrium dissociation constant (KD) of 12 nM or less, wherein the KD is measured using Proteon surface plasmon resonance assay ProteOn XPR36 system at +25° C.;
- bind residues 1-6 of CD3e as determined by X-ray crystallography; or
- activate T cells or induces CD69 expression to a similar degree as cOKT3 or SP34-2 as determined by fluorescence-activated cell sorting assay.

In some embodiments, the antibodies or antigen-binding fragments thereof described herein comprise the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2, and the LCDR3 of SEQ ID NOs:662, 663, 664, 671, 673, and 690, respectively; a VH and a VL of SEQ ID NOs:652 and 661, respectively; or a HC and a LC of SEQ ID NOs:640 and 676, respectively.

In some embodiments, the antibodies or antigen-binding fragments thereof described herein comprise the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2, and the LCDR3 of SEQ ID NOs:662, 663, 664, 773, 673, and 690, respectively; a VH and a VL of SEQ ID NOs:657 and 678, respectively or a HC and a LC of SEQ ID NOs:675 and 677, respectively.

Further described herein are bispecific antibodies comprising a first domain that specifically binds CD3 and a second domain that specifically binds a second antigen, wherein the first domain comprises an antibody or antigen-binding fragment thereof described herein.

The bispecific antibodies described herein can comprise a first domain that comprises the HCDR1, HCDR2, the HCDR3, the LCDR1, the LCDR2, and the LCDR3 of SEQ ID NOs:662, 663, 664, 671, 673, and 670, respectively; and a second domain that comprises the the HCDR1, HCDR2, the HCDR3, the LCDR1, the LCDR2, and the LCDR3 of SEQ ID NOs:697, 683, 698, 699, 792, and 686, respectively;

The bispecific antibodies described herein can comprise a first domain that comprises the VH and VL of SEQ ID NOs:652 and 661, respectively; and a second domain that comprises the VH and VL of SEQ ID NOs:681 and 682, respectively.

The bispecific antibodies described herein can comprise a first domain that comprises the HC and LC of SEQ ID NOs:640 and 676, respectively; and a second domain that comprises the HC and LC of SEQ ID NOs:679 and 680, respectively.

The disclosure further provides isolated bispecific CD3×PSMA antibodies comprising a first domain that binds to cells expressing recombinant Macaca fascicularis or human CD3d or CD3e with an affinity of 300 nM or less, wherein the binding to cells is measured by flow cytometry, and a second domain that specifically bind PSMA.

In some embodiments, the bispecific CD3×PSMA antibody comprises a first domain that:

- binds human and Macaca fascicularis CD3+T lymphocytes with a calculated EC50 of 20 nM or less and binds Macaca fascicularis CD3-expressing HEK cells with a calculated EC50 of 40 nM or less, wherein the difference in calculated EC50 between binding CD3+T lymphocytes and binding Macaca fascicularis CD3-expressing HEK cells is less than 5-fold, and wherein the calculated EC50 is measured in a whole cell binding assay at 0° C. using flow cytometry;
- binds recombinant CD3d from human (SEQ ID NO:691), or binds recombinant CD3e from human (SEQ ID NO:636), or binds recombinant CD3d from Macaca fascicularis (SEQ ID NO:692), or binds recombinant CD3e from Macaca fascicularis (SEQ ID NO:693) with an equilibrium dissociation constant (KD) of 12 nM or less, wherein the KD is measured using Proteon surface plasmon resonance assay ProteOn XPR36 system at +25° C.;
- displays no methionine or tryptophan oxidation, or displays no asparagine deamidation, or displays no asparagine isomerization as detected by peptide mapping analysis;
- binds residues 1-6 of CD3e as determined by X-ray crystallography; or
- activates T cells or induces CD69 expression to a similar degree as cOKT3 or SP34-2 as determined by fluorescence-activated cell sorting assay.

Also described are pharmaceutical compositions comprising the antibodies described herein and a pharmaceutically acceptable carrier.

The disclosure further provides methods of producing the antibodies described herein, comprising culturing a host cell comprising a vector comprising a polynucleotide encoding the antibodies or antigen-binding fragments thereof described herein in conditions that the antibody is expressed, and recovering the antibody produced by the host cell. The methods of producing the bispecific CD3×PSMA antibody can comprise combining a monospecific bivalent CD3 antibody having two identical HC1 and two identical LC1 and a monospecific bivalent PSMA antibody having two identical HC2 and two identical LC2 in a mixture of about 1:1 molar ratio; introducing a reducing agent into the mixture; incubating the mixture about ninety minutes to about six hours; removing the reducing agent; and purifying the bispecific CD3×PSMA antibody that comprises the HC1, the LC1, the HC2 and the LC2.

Further described are methods of treating a cancer in a subject, comprising administering a therapeutically effective amount of the isolated antibodies described to the subject in need thereof for a time sufficient to treat the cancer.

The disclosure also provides kits comprising the antibodies described herein. The kits can further comprise reagents for detecting the antibodies and instructions of use.

BRIEF DESCRIPTIONS OF THE DRAWINGS

The summary, as well as the following detailed description, is further understood when read in conjunction with the appended drawings. For the purpose of illustrating the disclosed antibodies and methods, there are shown in the drawings exemplary embodiments of the antibodies and methods; however, antibodies and methods are not limited to the specific embodiments disclosed. In the drawings:

FIGS. 1A and 1B show anti-CD3 antibodies produced in OmniRat. VH (FIG. 1A) and VL (FIG. 1B) sequences of active anti-CD3 mAbs produced in OmniRat were aligned with human germline sequences from IMGT. CDR regions are underlined. Sequence divergence is shown in bold. FIG. 1A discloses SEQ ID NOS 651, 651, 653, 656, 655, 20, 654 and 717 and FIG. 1B discloses SEQ ID NOS 658, 688, 660, 659, 659, 659, 659 and 718, all respectively, in order of appearance.

FIG. 2 shows a cell-based binding assay to assess the binding capacity of individual rat hybridoma supernatants to human purified CD3+T lymphocytes.

FIG. 3 shows a cell-based binding assay to assess the binding capacity of individual rat hybridoma supernatants to cynomolgus purified CD3+T lymphocytes.

FIG. 4 shows competition assay results of hybridoma supernatants assessed for their ability to compete with commercial anti-human CD3 antibody SP34-2, which has a known epitope and is cross-reactive with cynomolgus CD3.

FIG. 5 shows representative binding curves of anti-CD3 antibodies on primary human T cells.

FIG. 6 shows representative competition binding curves of anti-CD3 antibodies with AlexaFluor 488 conjugated SP34-2 on primary human T cells.

FIG. 7 shows binding of light chain (LC) engineered BLW-2E6 mAbs to primary human T cells.

FIG. 8 shows binding of heavy chain (HC) engineered BLW-2E6 to primary human T cells.

FIG. 9 shows AlexaFluor™ (AF) 488 CD3 Saturation in Human T Cells by FACS analysis. Acquired mean fluorescence intensity values were plotted as a function of the antibody molecule concentration. Kd values were derived for each donor, and the mean value obtained. The Saturation Binding Constant (KdT) for human T-Cells was derived to be 5.6±1.0 nM (n=4) and was used herein to determine Kd binding affinities. One human donor was excluded as it failed the viability criteria of being at least 60% during analysis. “LS” identifiers in figure legend refer to individual human T cell donors.

FIG. 10 shows inhibition curves for the bivalent anti CD3 antibodies, CD3B376 and CD3B450, for competing for binding against the AlexaFluor488 SP-34 anti CD3 antibody. IC50 values were derived to be 29 and 60 nM respectively.

FIG. 11 shows sensorgrams of BLW-2E6 variants binding to hCD3ε(1-27)-Tn25.

FIGS. 12A-12E shows thermal stability of anti-CD3 antibodies CD3B376 (FIG. 12A), CD3B450 (FIG. 12B), CD3B389 (FIG. 12C), CD3B467 by DSC (FIG. 12D), and overlay of the thermograms for all the candidates (FIG. 12E).

FIGS. 13A-13B show comparisons of the thermal stability of anti-CD3 antibodies overlay of thermograms for CD3B376 and CD3B389 (FIG. 13A); overlay of thermograms for CD3B450 and CD3B467 (FIG. 13B).

FIG. 14 shows LNCAP cell binding of a subset of affinity-matured PSMA×CD3 bispecific antibodies.

FIG. 15 shows LNCAP cell binding of a subset of affinity-matured PSMA×CD3 bispecific antibodies.

FIG. 16 shows PSMA-negative PC3 cell binding results of affinity-matured PSMA×CD3 bispecific antibodies.

FIG. 17 shows results of PSMA×CD3 Affinity Matured Bispecific Abs in a Functional Cell Killing Assay.

FIG. 18 shows antibody-antigen interactions in the CD3B334:CD3 complex. CD3 residues are in ellipses, CD3B334 residues are in boxes.

FIG. 19 shows a primary human and cynomolgus monkey T cell based assay used to determine the capacity of the hybridoma hits to activate T cells as measured by CD69 activation.

FIG. 20 shows anti-tumor efficacy of PS3B79 in LnCAP AR.TB human prostate xenografts in T cell humanized NSG mice. Subcutaneous LnCAP AR.TB tumors were measured twice weekly and the results presented as the mean tumor volume, expressed in mm³±SEM (*, p<0.0001).

FIG. 21 shows anti-tumor efficacy of PS3B90 in LnCAP AR.TB human prostate xenografts in T cell humanized NSG mice. Subcutaneous LnCAP AR.TB tumors were measured twice weekly and the results presented as the mean tumor volume, expressed in mm³±SEM (*, p<0.001).

FIGS. 22A-22D show titration curves for Anti-PSMA phage panning hits binding to human LNCaP cells. FIG. 22A shows titration curves for hits G9-PSM M18, M25, M50, M52, M56, M57, and M59; FIG. 22B shows M52 and M110; FIG. 22C shows M85, M87, and M81; and FIG. 22D shows M52 and M84. In FIG. 22D, Mammalian-expressed supernatants were normalized for Fab expression via octet, and titrated against either human LNCAP, PSMG5 (Cyno-PSMA HEK), or PSMG9 (chimp-PSMA HEK) cells using flowcytometry. Geometric mean fluorescence intensity (GeoMFI) was plotted vs. Fab concertation using GraphPad Prizm.

FIGS. 23A-23D show titration curves for Anti-PSMA phage panning hits binding to Chimpanzee-PSMA expressing HEK cells shows. (Mammalian Fab Supernatant Titration Curves for Anti-PSMA Phage Panning Hits vs Chimp-PSMA HEK). FIG. 23A shows titration curves for hits G9-PSM M18, M25, M50, M52, M56, M57, and M59; FIG. 23B shows M52 and M110; FIG. 23C shows M81, M52, M85, and M87; and FIG. 23D shows M52 and M84. In FIG. 23D, Mammalian-expressed supernatants were normalized for Fab expression via octet, and titrated against either human LNCAP, PSMG5 (Cyno-PSMA HEK), or PSMG9 (chimp-PSMA HEK) cells using flowcytometry. Geometric mean fluorescence intensity (GeoMFI) was plotted vs. Fab concertation using GraphPad Prizm.

FIGS. 24A-24D show titration curves for Anti-PSMA phage panning hits binding to Cynomolgus monkey PSMA-expressing HEK cells. (Mammalian Fab Supernatant Titration Curves for Anti-PSMA Phage Panning Hits vs Cyno PSMA HEK). FIG. 24A shows titration curves for hits G9-PSM M18, M25, M50, M52, M56, M57, and M59; FIG. 24B shows M52 and M110; FIG. 24C shows M81, M52, M85, and M87; and FIG. 24D shows M52 and M84. In FIG. 24D, Mammalian-expressed supernatants were normalized for Fab expression via octet, and titrated against either human LNCAP, PSMG5 (Cyno-PSMA HEK), or PSMG9 (chimp-PSMA HEK) cells using flow cytometry. Geometric mean fluorescence intensity (GeoMFI) was plotted vs. Fab concertation using GraphPad Prizm.

FIG. 25 shows the overall structure of PSMM84 Fab bound to human PSMA ECD homodimer.

FIG. 26 shows a close view of PSMA main interactions with the PSMB83 Light Chain.

FIG. 27 shows a close view of PSMA main interactions with the PSMB83 Heavy Chain

FIG. 28 shows the comparison of epitope residues of PSMB83 within the sequences of human (SEQ ID NO: 719), mouse (SEQ ID NO: 720) and cynomolgus monkey (cyno) (SEQ ID NO: 721) PSMA. Epitope residues are shaded and sequence divergence is shown by underline.

FIG. 29 shows the paratope residues of PSMB83. CDRs are underlined and paratope residues are shaded.

FIG. 30 discloses SEQ ID NOS 722-727, respectively, in order of appearance.

FIG. 30 shows an interaction map with direct contacts made between PSMA and PSMM84. Van der Waals interactions are shown as dashed lines and H-bonds are solid lines with arrows pointing to the backbone atoms.

FIG. 31 shows expression levels of anti-PSMA Fab clones derived from PSMM84 as compared to expression of parent PSMB83. Raw luminescence numbers were plotted against the log concentration.

FIG. 32 shows binding to human PSMA of anti-PSMA Fab clones derived from PSMB83 as compared to binding of parent PSMM84. Raw luminescence numbers were plotted against the log concentration.

FIG. 33 shows binding to cyno PSMA of anti-PSMA Fab clones derived from PSMB83 as compared to binding of parent PSMB83. Raw luminescence numbers were plotted against the log concentration.

FIG. 34 shows IC3B19 and IC3B34 Ex Vivo Mediated T cell Cytotoxicity of LAMA-84 cells in Whole Blood after 48 hours. The concentration of IC3B19 and IC3B34 is provided in the table in the lower part of the figure.

FIG. 35 shows IC3B19 and IC3B34 Ex Vivo Mediated T-cell Activation in Whole Blood after 48 hours. The concentration of IC3B19 and IC3B34 is provided in the table in the lower part of the figure.

FIGS. 36A-55B (descriptions below) show IC3B19 and IC3B34 mediated engagement of T cells and IL1RAP+ target cell line LAMA-84 (endogenous and exogenously added tumor cells). Supernatants were evaluated for 10 pro-inflammatory cytokines from whole blood (n=15 donors) cytotoxicity and T-cell activation assays with exogenous LAMA-84 IL1RAP+ tumor cell line added. For these figures, statistically significant differences are shown in bold text.

FIGS. 36A-36B show T cell IL-1beta release mediated by IC3B19 and IC3B34 (FIG. 36A) and corresponding 4PL regression parameter estimates (FIG. 36B) after 24 hours.

FIGS. 37A-37B show T cell IL-1beta release mediated by IC3B19 and IC3B34 (FIG. 37A) and corresponding 4PL regression parameter estimates (FIG. 37B) after 48 hours.

FIGS. 38A-38B show T cell IL-2 release mediated by IC3B19 and IC3B34 (FIG. 38A) and corresponding 4PL regression parameter estimates (FIG. 38B) after 24 hours.

FIGS. 39A-39B show T cell IL-2 release mediated by IC3B19 and IC3B34 (FIG. 39A) and corresponding 4PL regression parameter estimates (FIG. 39B) after 48 hours.

FIGS. 40A-40B show T cell IL-4 release mediated by IC3B19 and IC3B34 (FIG. 40A) and corresponding 4PL regression parameter estimates (FIG. 40B) after 24 hours.

FIG. 41 shows T cell IL-4 release mediated by IC3B19 and IC3B34 after 48 hours.

FIGS. 42A-42B show T cell IL-6 release mediated by IC3B19 and IC3B34 (FIG. 42A) and corresponding 4PL regression parameter estimates (FIG. 42B) after 24 hours.

FIG. 43 shows T cell IL-6 release mediated by IC3B19 and IC3B34 after 48 hours.

FIG. 44 shows T cell IL-8 release mediated by IC3B19 and IC3B34 after 24 hours.

FIG. 45 shows T cell IL-8 release mediated by IC3B19 and IC3B34 after 48 hours.

FIGS. 46A-46B show T cell IL-10 release mediated by IC3B19 and IC3B34 (FIG. 46A) and corresponding 4PL regression parameter estimates (FIG. 46B) after 24 hours.

FIGS. 47A-47B show T cell IL-10 release mediated by IC3B19 and IC3B34 (FIG. 47A) and corresponding 4PL regression parameter estimates (FIG. 47B) after 48 hours.

FIG. 48 shows T cell IL-12p70 release mediated by IC3B19 and IC3B34 after 24 hours.

FIG. 49 shows T cell IL-12p70 release mediated by IC3B19 and IC3B34 after 48 hours.

FIG. 50 shows T cell IL-13 release mediated by IC3B19 and IC3B34 after 24 hours.

FIGS. 51A-51B show T cell IL-13 release mediated by IC3B19 and IC3B34 (FIG. 51A) and corresponding 4PL regression parameter estimates (FIG. 51B) after 48 hours.

FIGS. 52A-52B show T cell IFN-gamma release mediated by IC3B19 and IC3B34 (FIG. 52A) and corresponding 4PL regression parameter estimates (FIG. 52B) after 24 hours.

FIGS. 53A-53B show T cell IFN-gamma release mediated by IC3B19 and IC3B34 (FIG. 53A) and corresponding 4PL regression parameter estimates (FIG. 53B) after 48 hours.

FIGS. 54A-54B show T cell TNF-alpha release mediated by IC3B19 and IC3B34 (FIG. 54A) and corresponding 4PL regression parameter estimates (FIG. 54B) after 24 hours.

FIGS. 55A-55B show T cell TNF-alpha release mediated by IC3B19 and IC3B34 (FIG. 55A) and corresponding 4PL regression parameter estimates (FIG. 55B) after 48 hours.

FIG. 56 shows IC3B19 and IC3B34, but not the bispecific antibodies with null arms (IAPB57×B23B49 or B23B39×CD3B219), induced target specific cytotoxicity in NCI-H1975 cells. In this assay, the cytotoxicity EC50s vary three-fold between IC3B19 and IC3B34, with values of 0.018 and 0.057 nM, respectively.

FIG. 57 shows Anti-Tumor Efficacy of IAPB57×CD3B376 in H1975 Human NSCLC Xenografts in T cell Humanized NSG Mice. Subcutaneous H1975 tumors were measured twice weekly and the results presented as the average tumor volume, expressed in mm3±SEM, *p≤0.0001.

FIG. 58 shows a comparison of isoelectric points for various anti-PSMA constructs.

FIG. 59 plots change in wavelength compared to CNTO5825 control molecule. Control CNTO607 shows characteristically strong self-interaction. Error bars represent standard deviations from triplicate measurements.

FIG. 60 shows a comparison of retention times on IgG and control columns in an anti-PSMA cross-interaction assay.

FIGS. 61A-61B show ex vivo assessment of CD33×CD3 bispecific antibodies using anti-CD3 arm CD3B219 and CD3B376 cytotoxicity of blasts and T cell activation in fresh AML patient whole blood. FIG. 61A shows the percent of total cell cytotoxicity of AML cells using CD33 bispecific antibodies or the CD3×null controls. FIG. 61B shows T cell activation induced by CD33 bispecific antibodies or the CD3×null controls. No Fc blocker was added.

FIGS. 62A-62C show CD33×CD3 T-cell mediated cytotoxicity assays. CD33×CD3 bispecific antibodies using anti-CD3 arm CD3B219 and anti-CD3B376 were incubated with human pan T cells and AML cell lines that are either wildtype (KG1, FIG. 62A), heterozygous (SH2, FIG. 62B) or homozygous (OCIAML3, FIG. 62C) for the CD33 SNP rs12459419 mutation. After 48 hr at 37° C., 5% CO2, total tumor cell cytotoxicity was measured by flow cytometry.

FIGS. 63A-63B show ex vivo assessment of C33B904 antibodies paired with either CD3B219 or CD3B376 on the cytotoxicity of MOLM-13 cells exogenously added to normal healthy human whole blood (N=6 donors): Percent of cytotoxicity of MOLM-13 cells (FIG. 63A) and CD33⁺CD14⁺ monocytes (FIG. 63B) using CD33×CD3 bispecifics and respective null×CD3 controls at 48 hrs.

FIGS. 64A-64B show ex vivo assessment of CD33×CD3 bispecific antibodies using anti-CD3 arm CD3B219 and CD3B376 on the cytotoxicity of monocytes and T cell activation in fresh whole blood from six normal cynomologous monkey donors. FIG. 64A shows the percent of total cell cytotoxicity of CD33⁺CD14⁺ cyno monocytes using CD33 bispecific antibodies or their CD3×null controls. FIG. 64B shows T cell activation induced by CD33 bispecific antibodies or their CD3×null controls. No Fc blocker was added.

FIG. 65 shows anti-tumor efficacy of C3CB189 in MOLM-13 human AML xenografts in T cell humanized NSG mice. Disseminated MOLM-13 tumors were imaged for bioluminescence (BLI) twice weekly and the results presented as average radiance (p/s/cm²/sr)±SEM (n=8-10/group). *p≤0.0001 for treatment vs. control, calculated by two-way ANOVA with Bonferroni test.

FIG. 66 shows survival of animals treated with C3CB189 in MOLM-13 Human AML xenografts in T cell humanized NSG mice. Survival of MOLM-13 bearing mice is graphically represented using a Kaplan-Meier curve and evaluated by Log-rank (Mantel-Cox) test. *p≤0.0001 for treatment vs. control groups.

FIG. 67 shows anti-tumor efficacy of C3CB88 in MOLM-13 human AML xenografts in T cell humanized NSG mice. Disseminated MOLM-13 tumors were imaged for bioluminescence (BLI) twice weekly and the results presented as average radiance (p/s/cm²/sr)±SEM (n=8-10/group). *p≤0.0001 for treatment vs. control, calculated by two-way ANOVA with Bonferroni test.

FIG. 68 shows survival of animals treated with C3CB88 in MOLM-13 human AML xenografts in T cell humanized NSG mice. Survival of MOLM-13 bearing mice is graphically represented using a Kaplan-Meier curve and evaluated by Log-rank (Mantel-Cox) test. *p≤0.05 for treatment vs. control groups.

FIG. 69 shows the alignment of select anti-TMEFF2 antibody heavy chain variable regions (VH). The VH regions are identified by their SEQ ID NO: at the beginning of each row.

FIG. 70 shows the alignment of select anti-TMEFF2 antibody light chain variable regions (VL). The VH regions are identified by their SEQ ID NO: at the beginning of each row.

FIG. 71 shows the reduction in mean tumor volume of each mouse treated with 0.5 mg/kg TMCB132 in an ex vivo LnCaP prostate cancer model in male NGS mice.

FIG. 72 shows efficacy of TMEB762×CD3B376 in established LNCaP Xenografts in T Cell Humanized NSG Mice.

FIG. 73 shows T-cell activation in LnCaP prostate cancer cells in response to administration of TMCB132.

FIG. 74 shows T cell-mediated cytotoxicity of TMCB132.

FIG. 75 shows the antitumor efficacy of TMCB132 in T-cell humanized mice.

DETAILED DESCRIPTION OF THE INVENTION

All publications, including but not limited to patents and patent applications, cited in this specification are herein incorporated by reference as though fully set forth.

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. Unless defined otherwise, 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 invention pertains.

Although any methods and materials similar or equivalent to those described herein may be used in the practice for testing of the present invention, exemplary materials and methods are described herein. In describing and claiming the present invention, the following terminology will be used.

As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to “a cell” includes a combination of two or more cells, and the like.

“Specific binding” or “specifically binds” or “binds” refers to an antibody binding to an antigen or an epitope within the antigen with greater affinity than for other antigens. Typically, the antibody binds to the antigen or the epitope within the antigen with an equilibrium dissociation constant (K_D) of about 5×10⁻⁸M or less, for example about 1×10⁻⁹M or less, about 1×10⁻¹⁰M or less, about 1×10⁻¹¹M or less, or about 1×10⁻²M or less, typically with the K_Dthat is at least one hundred-fold less than its K_Dfor binding to a non-specific antigen (e.g., BSA, casein). The dissociation constant may be measured using standard procedures. Antibodies that specifically bind to the antigen or the epitope within the antigen may, however, have cross-reactivity to other related antigens, for example to the same antigen from other species (homologs), such as human or monkey, for example Macaca fascicularis (cynomolgus, cyno) or Pan troglodytes (chimpanzee, chimp). While a monospecific antibody specifically binds one antigen or one epitope, a bispecific antibody specifically binds two distinct antigens or two distinct epitopes.

“Antibodies” is meant in a broad sense and includes immunoglobulin molecules including monoclonal antibodies including murine, human, humanized and chimeric monoclonal antibodies, antigen binding fragments, bispecific or multispecific antibodies, dimeric, tetrameric or multimeric antibodies, single chain antibodies, domain antibodies and any other modified configuration of the immunoglobulin molecule that comprises an antigen binding site of the required specificity. “Full length antibody molecules” are comprised of two heavy chains (HC) and two light chains (LC) inter-connected by disulfide bonds as well as multimers thereof (e.g. IgM). Each heavy chain is comprised of a heavy chain variable region (VH) and a heavy chain constant region (comprised of domains CH1, hinge, CH2 and CH3). Each light chain is comprised of a light chain variable region (VL) and a light chain constant region (CL). The VH and the VL regions may be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with framework regions (FR). Each VH and VL is composed of three CDRs and four FR segments, arranged from amino-to-carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4.

“Complementarity determining regions (CDR)” are antibody regions that bind an antigen. CDRs may be defined using various delineations such as Kabat (Wu et al. (1970) J Exp Med 132: 211-50) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), Chothia (Chothia et al. (1987) J Mol Biol 196: 901-17), IMGT (Lefranc et al. (2003) Dev Comp Immunol 27: 55-77) and AbM (Martin and Thornton (1996) J Bmol Biol 263: 800-15). The correspondence between the various delineations and variable region numbering are described (see e.g. Lefranc et al. (2003) Dev Comp Immunol 27: 55-77; Honegger and Pluckthun, (2001) J Mol Biol 309:657-70; International ImMunoGeneTics (IMGT) database; Web resources, http://www_imgt_org). Available programs such as abYsis by UCL Business PLC may be used to delineate CDRs. The term “CDR”, “HCDR1”, “HCDR2”, “HCDR3”, “LCDR1”, “LCDR2” and “LCDR3” as used herein includes CDRs defined by any of the methods described supra, Kabat, Chothia, IMGT or AbM, unless otherwise explicitly stated in the specification.

Immunoglobulins may be assigned to five major classes, IgA, IgD, IgE, IgG and IgM, depending on the heavy chain constant domain amino acid sequence. IgA and IgG are further sub-classified as the isotypes IgA1, IgA2, IgG1, IgG2, IgG3 and IgG4. Antibody light chains of any vertebrate species may be assigned to one of two clearly distinct types, namely kappa (κ) and lambda (λ), based on the amino acid sequences of their constant domains.

“Antigen binding fragment” refers to a portion of an immunoglobulin molecule that binds an antigen. Antigen binding fragments may be synthetic, enzymatically obtainable or genetically engineered polypeptides and include the VH, the VL, the VH and the VL, Fab, F(ab′)2, Fd and Fv fragments, domain antibodies (dAb) consisting of one VH domain or one VL domain, shark variable IgNAR domains, camelized VH domains, minimal recognition units consisting of the amino acid residues that mimic the CDRs of an antibody, such as FR3-CDR3-FR4 portions, the HCDR1, the HCDR2 and/or the HCDR3 and the LCDR1, the LCDR2 and/or the LCDR3. VH and VL domains may be linked together via a synthetic linker to form various types of single chain antibody designs where the VH/VL domains may pair intramolecularly, or intermolecularly in those cases when the VH and VL domains are expressed by separate single chain antibody constructs, to form a monovalent antigen binding site, such as single chain Fv (scFv) or diabody; described for example in Int. Patent Publ. Nos. WO1998/44001, WO1988/01649, WO1994/13804 and WO1992/01047.

“Monoclonal antibody” refers to an antibody obtained from a substantially homogenous population of antibody molecules, i.e., the individual antibodies comprising the population are identical except for possible well-known alterations such as removal of C-terminal lysine from the antibody heavy chain or post-translational modifications such as amino acid isomerization or deamidation, methionine oxidation or asparagine or glutamine deamidation. Monoclonal antibodies typically bind one antigenic epitope. A bispecific monoclonal antibody binds two distinct antigenic epitopes. Monoclonal antibodies may have heterogeneous glycosylation within the antibody population. Monoclonal antibody may be monospecific or multispecific such as bispecific, monovalent, bivalent or multivalent.

“Isolated antibody” refers to an antibody or antibody fragment that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody specifically binding an antigen is substantially free of antibodies that specifically bind antigens other than the antigen). In the case of bispecific CD3 antibodies, the bispecific antibody specifically binds both CD3 a second antigen. “Isolated antibody” encompasses antibodies that are isolated to a higher purity, such as antibodies that are 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% pure.

“Humanized antibody” refers to an antibody in which at least one CDR is derived from non-human species and at least one framework is derived from human immunoglobulin sequences. Humanized antibody may include substitutions in the frameworks so that the frameworks may not be exact copies of expressed human immunoglobulin or human immunoglobulin germline gene sequences.

“Human antibody” refers to an antibody that is optimized to have minimal immune response when administered to a human subject. Variable regions of human antibody are derived from human immunoglobulin sequences. If human antibody contains a constant region or a portion of the constant region, the constant region is also derived from human immunoglobulin sequences. Human antibody comprises heavy and light chain variable regions that are “derived from” sequences of human origin if the variable regions of the human antibody are obtained from a system that uses human germline immunoglobulin or rearranged immunoglobulin genes. Such exemplary systems are human immunoglobulin gene libraries displayed on phage, and transgenic non-human animals such as mice or rats carrying human immunoglobulin loci. “Human antibody” typically contains amino acid differences when compared to the immunoglobulins expressed in humans due to differences between the systems used to obtain the human antibody and human immunoglobulin loci, introduction of somatic mutations or intentional introduction of substitutions into the frameworks or CDRs, or both. Typically, “human antibody” is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical in amino acid sequence to an amino acid sequence encoded by human germline immunoglobulin or rearranged immunoglobulin genes. In some cases, “human antibody” may contain consensus framework sequences derived from human framework sequence analyses, for example as described in Knappik et al., (2000) J Mol Biol 296:57-86, or synthetic HCDR3 incorporated into human immunoglobulin gene libraries displayed on phage, for example as described in Shi et al., (2010) J Mol Biol 397:385-96, and in Int. Patent Publ. No. WO2009/085462.

“Percent (%) amino acid sequence identity” with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For purposes herein, however, % amino acid sequence identify values are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was authored by Genentech. Inc., and the source code has been filed with user documentation in the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech. Inc., South San Francisco, Calif., or may be compiled from the source code. The ALIGN-2 program should be compiled for use on a UNIX operating system, including digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary. Where ALIGN-2 is employed for amino acid sequence comparisons, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows:

100 times the fraction X/Y,

where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in that program's alignment of A and B, and where Y is the total number of amino acid residues in B. It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A. Unless specifically stated otherwise, all % amino acid sequence identity values used herein are obtained as described in the immediately preceding paragraph using the ALIGN-2 computer program.

Antibodies in which antigen binding sites are derived from a non-human species are not included in the definition of “human antibody”.

“Recombinant” refers to DNA, antibodies and other proteins that are prepared, expressed, created or isolated by recombinant means when segments from different sources are joined to produce recombinant DNA, antibodies or proteins.

“Epitope” refers to a portion of an antigen to which an antibody specifically binds. Epitopes typically consist of chemically active (such as polar, non-polar or hydrophobic) surface groupings of moieties such as amino acids or polysaccharide side chains and may have specific three-dimensional structural characteristics, as well as specific charge characteristics. An epitope may be composed of contiguous and/or discontiguous amino acids that form a conformational spatial unit. For a discontiguous epitope, amino acids from differing portions of the linear sequence of the antigen come in close proximity in 3-dimensional space through the folding of the protein molecule. Antibody “epitope” depends on the methodology used to identify the epitope.

“Paratope” refers to a portion of an antibody to which an antigen specifically binds. A paratope may be linear in nature or may be discontinuous, formed by a spatial relationship between non-contiguous amino acids of an antibody rather than a linear series of amino acids. A “light chain paratope” and a “heavy chain paratope” or “light chain paratope amino acid residues” and “heavy chain paratope amino acid residues” refer to antibody light chain and heavy chain residues in contact with an antigen, respectively, or in general, “antibody paratope residues” refer to those antibody amino acids that are in contact with antigen.

“Bispecific” refers to an antibody that specifically binds two distinct antigens or two distinct epitopes within the same antigen. The bispecific antibody may have cross-reactivity to other related antigens, for example to the same antigen from other species (homologs), such as human or monkey, for example Macaca fascicularis (cynomolgus, cyno) or Pan troglodytes, or may bind an epitope that is shared between two or more distinct antigens.

“Multispecific” refers to an antibody that specifically binds two or more distinct antigens or two or more distinct epitopes within the same antigen. The multispecific antibody may have cross-reactivity to other related antigens, for example to the same antigen from other species (homologs), such as human or monkey, for example Macaca fascicularis (cynomolgus, cyno) or Pan troglodytes, or may bind an epitope that is shared between two or more distinct antigens.

“Variant” refers to a polypeptide or a polynucleotide that differs from a reference polypeptide or a reference polynucleotide by one or more modifications, for example one or more substitutions, insertions or deletions.

“Vector” refers to a polynucleotide capable of being duplicated within a biological system or that can be moved between such systems. Vector polynucleotides typically contain elements, such as origins of replication, promoter, polyadenylation signal and selection markers, that function to facilitate the duplication or maintenance of these polynucleotides in a biological system, such as a cell, virus, animal, plant, and reconstituted biological systems utilizing biological components capable of duplicating a vector. The vector polynucleotide may be DNA or RNA molecules or a hybrid of these, single stranded or double stranded.

“Expression vector” refers to a vector that can be utilized in a biological system or in a reconstituted biological system to direct the translation of a polypeptide encoded by a polynucleotide sequence present in the expression vector.

“Polynucleotide” refers to a molecule comprising a chain of nucleotides covalently linked by a sugar-phosphate backbone or other equivalent covalent chemistry. Double and single-stranded DNA and RNA are typical examples of polynucleotides. “Polynucleotide” may refer to a synthetic molecule comprising a chain of nucleotides covalently linked by a sugar-phosphate backbone or other equivalent covalent chemistry. cDNA is an exemplary synthetic polynucleotide.

“Polypeptide” or “protein” refers to a molecule that comprises at least two amino acid residues linked by a peptide bond to form a polypeptide. Small polypeptides of less than 50 amino acids may be referred to as “peptides”.

“Flow cytometry” is a technology that is used to analyze the physical and chemical characteristics of particles in a fluid as it passes through at least one laser. Cell components are fluorescently labelled and then excited by the laser to emit light at varying wavelengths (Adan, et al, Critical Reviews in Biotechnology (2016) 1549-7801).

“Anti-idiotypic (anti-Id) antibody” is an antibody that recognizes the antigenic determinants (e.g. the paratope or CDRs) of the antibody. It is generally known in the art the process of producing or preparing an anti-idiotypic antibody. (Lathey, J. et al Immunology 1986 57(1):29-35). The anti-Id antibody may be antigen-blocking or non-blocking. The antigen-blocking anti-Id antibody may be used to detect the free antibody in a sample, e.g. CD3. The non-blocking anti-Id antibody may be used to detect the total antibody (free, partially bound to antigen, or fully bound to antigen) in a sample. An anti-Id antibody may be prepared by immunizing an animal with the antibody to which an anti-Id antibody is being prepared. In some embodiments described herein, the anti-idiotypic antibody is used for detecting the level of the therapeutic antibodies in a sample.

An anti-Id antibody may also be used as an immunogen to induce an immune response in yet another animal, producing a so-called anti-anti-Id antibody. An anti-anti-Id antibody may be epitopically identical to the original mAb, which induced the anti-Id antibody. Thus, by using antibodies to the idiotypic determinants of a mAb, it is possible to identify other clones expressing antibodies of identical specificity. Anti-Id antibodies may be varied (thereby producing anti-Id antibody variants) and/or derivatized by any suitable technique, such as those described elsewhere herein with respect to the anti-CD3 antibodies.

PSMA refers to Prostate Specific Membrane Antigen. The amino acid sequence of Pan troglodytes (also referred to as chimpanzee or chimp) PSMA is shown in SEQ ID NO: 49. The extracellular domain spans residues 44-750, the transmembrane domain spans residues 20-43 and the cytoplasmic domain spans residues 1-19 of SEQ ID NO: 49. The amino acid sequence of the Macaca fascicularis (also referred to as cynomolgus monkey, macaque or cyno) PSMA is shown in SEQ ID NO: 50. The extracellular domain spans residues 44-750, the transmembrane domain spans residues 20-43 and the cytoplasmic domain spans residues 1-19 of SEQ ID NO: 50. The amino acid sequence of the human PSMA is shown in SEQ ID NO:51. The extracellular domain spans residues 44-750, the transmembrane domain spans residues 20-43 and the cytoplasmic domain spans residues 1-19 of SEQ ID NO:51.

Throughout the specification, “CD3-specific” or “specifically binds CD3” or “anti-CD3 antibody” refers to antibodies that bind specifically to the CD3-epsilon polypeptide (SEQ ID NO:635), including antibodies that bind specifically to the CD3-epsilon extracellular domain (ECD) (SEQ ID NO:636). CD3-epsilon, together with CD3-gamma, -delta and -zeta, and the T-cell receptor alpha/beta and gamma/delta heterodimers, forms the T-cell receptor-CD3 complex. This complex plays an important role in coupling antigen recognition to several intracellular signal-transduction pathways. The CD3 complex mediates signal transduction, resulting in T cell activation and proliferation. CD3 is required for the immune response.

(Human CD3 epsilon)

SEQ ID NO: 635

MQSGTHWRVLGLCLLSVGVWGQDGNEEMGGITQTPYKVSISGTTVILTCP

QYPGSEILWQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYP

RGSKPEDANFYLYLRARVCENCMEMDVMSVATIVIVDICITGGLLLLVYY

WSKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYS

GLNQRRI

(Human CD3 epsilon extracellular domain)

SEQ ID NO: 636

DGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDD

KNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENC

MEMD

As used herein, the terms “interleukin-1 receptor accessory protein”, “IL1RAP” and “IL1-RAP” specifically include the human IL1RAP protein, for example as described in GenBank Accession No. AAB84059, NCBI Reference Sequence: NP_002173.1 and UniProtKB/Swiss-Prot Accession No. Q9NPH3-1 (see also Huang et al., 1997, Proc. Natl. Acad. Sci. USA. 94 (24), 12829-12832). IL1RAP is also known in the scientific literature as IL1 R3, C3orf13, FLJ37788, IL-1 RAcP and EG3556.

“CD33” refers to cluster of differentiation 33 protein. CD33 is a 67 kilodalton (kD) single pass transmembrane glycoprotein and is a member of the sialic acid-binding immunoglobulin-like lectins (Siglecs) family. CD33 is primarily considered to be a myeloid differentiation antigen, with low expression in myeloid progenitors, neutrophils and macrophages and high expression in circulating monocytes and dendritic cells. Human CD33 extracellular domain (Uniprot P20138) (SEQ ID NO:636) and cynomolgus CD33 (XP_005590138.1) are examples of proteins for use in generating CD33-specific antibodies according to the present disclosure.

“TMEFF2” refers to human transmembrane protein with EGF like and two follistatin like domains 2, also called tomoregulin 2. The amino acid sequence of the full length human TMEFF2 is shown in SEQ ID NO: 77. The extracellular domain of TMEFF2 is shown in SEQ ID NO: 575 and spans residues 40-374 of the full length TMEFF2. TMEFF2 extracellular domain harbors three distinct subdomains, the Kazal-like 1 (residues 85-137), the Kazal-like 2 (residues 176-229) and the EGF domain (residues 261-301). The TMEFF2 EGF domain is shown in SEQ ID NO: 577. The TMEFF2 “membrane proximal region” refers to the TMEFF2 region of SEQ ID NO: 629, which encompasses the EGF domain and the N—C-terminal linker regions (e.g. residues 230-320 of full length human TMEFF2 of SEQ ID NO: 77). All references to proteins, polypeptides and protein fragments herein are intended to refer to the human version of the respective protein, polypeptide or protein fragment unless explicitly specified as being from a non-human species. Thus, “TMEFF2” means human TMEFF2 unless specified as being from a non-human species, e.g., “mouse TMEFF2” or “monkey TMEFF2” etc.

(full length human TMEFF2)

SEQ ID NO: 77

MVLWESPRQCSSWTLCEGFCWLLLLPVMLLIVARPVKLAAFPTSLSDCQT

PTGWNCSGYDDRENDLFLCDTNTCKFDGECLRIGDTVTCVCQFKCNNDYV

PVCGSNGESYQNECYLRQAACKQQSEILVVSEGSCATDAGSGSGDGVHEG

SGETSQKETSTCDICQFGAECDEDAEDVWCVCNIDCSQTNFNPLCASDGK

SYDNACQIKEASCQKQEKIEVMSLGRCQDNTTTTTKSEDGHYARTDYAEN

ANKLEESAREHHIPCPEHYNGFCMHGKCEHSINMQEPSCRCDAGYTGQHC

EKKDYSVLYVVPGPVRFQYVLIAAVIGTIQIAVICVVVLCITRKCPRSNR

IHRQKQNTGHYSSDNTTRASTRLI

(extracellular domain of human TMEFF2)

SEQ ID NO: 575

FPTSLSDCQTPTGWNCSGYDDRENDLFLCDTNTCKFDGECLRIGDTVTCV

CQFKCNNDYVPVCGSNGESYQNECYLRQAACKQQSEILVVSEGSCATDAG

SGSGDGVHEGSGETSQKETSTCDICQFGAECDEDAEDVWCVCNIDCSQTN

FNPLCASDGKSYDNACQIKEASCQKQEKIEVMSLGRCQDNTTTTTKSEDG

HYARTDYAENANKLEESAREHHIPCPEHYNGECMHGKCEHSINMQEPSCR

CDAGYTGQHCEKKDYSVLYVVPGPVRFQYVLIAAVIGTIQIAVICVVVLC

ITRKCPRSNRIHRQKQNTGHYSSDNTTRASTRLI

TMEFF2 EGF domain

SEQ ID NO: 577

HHIPCPEHYNGFCMHGKCEHSINMQEPSCRCDAGYTGQHCE

TMEFF2 membrane proximal region

SEQ ID NO: 629

NTTTTTKSEDGHYARTDYAENANKLEESAREHHIPCPEHYNGFCMHGKCE

HSINMQEPSCRCDAGYTGQHCEKKDYSVLYVVPGPVRFQYV

“TMEFF2 positive cancer” refers to a cancer tissue or a cancer cell that displays measurable level of TMEFF2 protein. Level of TMEFF2 protein may be measured using well known assays using, for example ELISA, immunofluorescence, flow cytometry or radioimmunoassay on live or lysed cells. “Overexpress”, “overexpressed” and “overexpressing” interchangeably refers to a sample such as a cancer cell, malignant cell or cancer tissue that has measurably higher levels of tumor antigen when compared to a reference sample. The overexpression may be caused by gene amplification or by increased transcription or translation. Expression and overexpression of protein in the sample may be measured using well-known assays using, for example ELISA, immunofluorescence, flow cytometry or radioimmunoassay on live or lysed cells. Expression and overexpression of a polynucleotide in the sample may be measured, for example, using fluorescent in situ hybridization, Southern blotting, or PCR techniques. A protein or a polynucleotide is overexpressed when the level of the protein or the polynucleotide in the sample is at least 1.5-fold higher when compared to the reference sample. Selection of the reference sample is well known.

“Sample” refers to a collection of similar fluids, cells, or tissues isolated from a subject, as well as fluids, cells, or tissues present within a subject. Exemplary samples are biological fluids such as blood, serum and serosal fluids, plasma, lymph, urine, saliva, cystic fluid, tear drops, feces, sputum, mucosal secretions of the secretory tissues and organs, vaginal secretions, ascites fluids such as those associated with non-solid tumors, fluids of the pleural, pericardial, peritoneal, abdominal and other body cavities, fluids collected by bronchial lavage, liquid solutions contacted with a subject or biological source, for example, cell and organ culture medium including cell or organ conditioned medium, lavage fluids and the like, tissue biopsies, fine needle aspirations or surgically resected tumor tissue.

A “cancer cell” or a “tumor cell” as used herein refers to a cancerous, pre-cancerous or transformed cell, either in vivo, ex vivo, or in tissue culture, that has spontaneous or induced phenotypic changes. These changes do not necessarily involve the uptake of new genetic material. Although transformation may arise from infection with a transforming virus and incorporation of new genomic nucleic acid or uptake of exogenous nucleic acid, it can also arise spontaneously or following exposure to a carcinogen, thereby mutating an endogenous gene. Transformation/cancer is exemplified by morphological changes, immortalization of cells, aberrant growth control, foci formation, proliferation, malignancy, modulation of tumor specific marker levels, invasiveness, tumor growth in suitable animal hosts such as nude mice, and the like, in vitro, in vivo, and ex vivo (Freshney, Culture of Animal Cells: A Manual of Basic Technique (3rd ed. 1994)).

Unless otherwise stated, any numerical values, such as a concentration or a concentration range described herein, are to be understood as being modified in all instances by the term “about.” Thus, a numerical value typically includes ±10% of the recited value. For example, a concentration of 1 mg/mL includes 0.9 mg/mL to 1.1 mg/mL. Likewise, a concentration range of 1% to 10% (w/v) includes 0.9% (w/v) to 11% (w/v). As used herein, the use of a numerical range expressly includes all possible subranges, all individual numerical values within that range, including integers within such ranges and fractions of the values unless the context clearly indicates otherwise.

“Effector antigens” are antigens from cells of the immune system that can stimulate or trigger cytotoxicity, phagocytosis, antigen presentation, and/or cytokine release. Such effector antigens are from, for example but not limited to, T cells and natural killer (NK) cells. Examples of suitable specificities for effector antigens include but are not limited to CD3 or CD3 subunits such as CD3ε for T cells and CD16 for NK cells. Such cell surface molecules of effector cells are suitable for mediating cell killing. Effector cells are cells of the immune system that can stimulate or trigger cytotoxicity, phagocytosis, antigen presentation, and/or cytokine release. Such effector cells are, for example but not limited to, T-cells, natural killer (NK) cells, granulocytes, monocytes, macrophages, dendritic cells, and antigen-presenting cells. Examples of suitable specificities for effector cells include but are not limited to CD2, CD3 and CD3 subunits such as CD3e, CD5, CD28 and other components of the T-cell receptor (TCR) for T cells; CD16, CD16A, CD25, CD38, CD44, CD56, CD69, CD94, CD335 (NKp46), CD336, (NKp44), CD337 (NKp30), NKp80, NKG2C and NKG2D, DNAM, NCRs for NK cells; CD18, CD64 and CD89 for granulocytes; CD18, CD32, CD64, CD89 and mannose receptor for monocytes and macrophages; CD64 and mannose receptor for dendritic cells; as well as CD35. In certain embodiments of the inventions, those specificities, i.e. cell surface molecules, of effector cells are suitable for mediating cell killing upon binding of a bispecific or multispecific molecules to such cell surface molecule and, thereby, inducing cytolysis or apoptosis.

“Bispecific CD3 antibody” refers to a molecule comprising at least one binding domain specifically binding CD3 and at least one binding domain specifically binding a second antigen, such as a bispecific antibody comprising a first domain that specifically binds CD3 and a second domain that specifically binds a second antigen. The domains specifically binding CD3 and a second antigen are typically V_H/V_Lpairs. The bispecific CD3 antibody may be monovalent in terms of its binding to either CD3 or the second antigen. In some embodiments, the second, or target, antigen is a cell surface antigen that is expressed on a target cell other than an immune effector cell. In some embodiments, the second antigen a tumor associated antigen (TAA). Exemplary TAAs are PSMA, CD33, IL1RAP, and TMEFF2.

“Bispecific PSMA×CD3 antibody”, “PSMA/CD3 antibody”, “bispecific anti-PSMA×CD3 antibody” or “anti-PSMA/CD3 antibody” and the like refer to a molecule comprising at least one binding domain specifically binding PSMA and at least one binding domain specifically binding CD3. The domains specifically binding PSMA and CD3 are typically VH/VL pairs. The bispecific anti-PSMA×CD3 antibody may be monovalent in terms of its binding to either PSMA or CD3.

“Bispecific CD33×CD3 antibody”, “CD33/CD3 antibody”, “bispecific anti-CD33×CD3 antibody” or “anti-CD33/CD3 antibody” and the like refer to a molecule comprising at least one binding domain specifically binding CD33 and at least one binding domain specifically binding CD3. The domains specifically binding CD33 and CD3 are typically V_H/V_Lpairs. The bispecific anti-CD33×CD3 antibody may be monovalent in terms of its binding to either CD33 or CD3.

“Bispecific IL1RAP×CD3 antibody”, “IL1RAP/CD3 antibody”, “bispecific anti-IL1RAP×CD3 antibody” or “anti-IL1RAP/CD3 antibody” and the like refer to a molecule comprising at least one binding domain specifically binding IL1RAP and at least one binding domain specifically binding CD3. The domains specifically binding IL1RAP and CD3 are typically V_H/V_Lpairs. The bispecific anti-IL1RAP×CD3 antibody may be monovalent in terms of its binding to either IL1RAP or CD3.

“Bispecific anti-TMEFF2/anti-CD3 antibody”, TMEFF2/CD3 antibody, TMEFF2×CD3 antibody and the like refer to an antibody that binds TMEFF2 and CD3.

“Valent” refers to the presence of a specified number of binding sites specific for an antigen in a molecule. As such, the terms “monovalent”, “bivalent”, “tetravalent”, and “hexavalent” refer to the presence of one, two, four and six binding sites, respectively, specific for an antigen in a molecule. “Multivalent” refers to the presence of two or more binding sites specific for an antigen in a molecule.

“An antigen specific CD4+ or CD8+ T cell” refers to a CD4+ or CD8+ T cell activated by a specific antigen, or immunostimulatory epitope thereof.

“Subject” includes any human or nonhuman animal. “Nonhuman animal” includes all vertebrates, e.g., mammals and non-mammals, such as nonhuman primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc. Except when noted, the terms “patient” or “subject” are used interchangeably.

The numbering of amino acid residues in the antibody constant region throughout the specification is according to the EU index as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991), unless otherwise explicitly stated.

TABLE 1

Conventional one- and three-letter amino acid codes used herein

Amino acid
Three-letter code
One-letter code

Alanine
Ala
A

Arginine
Arg
R

Asparagine
Asn
N

Aspartate
Asp
D

Cysteine
Cys
C

Glutamate
Gln
E

Glutamine
Glu
Q

Glycine
Gly
G

Histidine
His
H

Isoleucine
Ile
I

Lysine
Lys
K

Methionine
Met
M

Phenylalanine
Phe
F

Proline
Pro
P

Serine
Ser
S

Threonine
Thr
T

Tryptophan
Trp
W

Tyrosine
Tyr
Y

Valine
Val
V

Compositions of Matter

The present invention provides anti-CD3 antibodies and antigen-binding fragments thereof, antibodies and antigen-binding fragments thereof that specifically bind PSMA, antibodies and antigen-binding fragments thereof that specifically bind CD33, antibodies and antigen-binding fragments thereof that specifically bind IL1RAP, multispecific antibodies comprising a first domain that specifically binds CD3 and a second domain that specifically binds a second antigen, and multispecific antibodies that specifically bind CD3 and one or more of PSMA, CD33, IL1RAP, and TMEFF2. The present invention provides polypeptides and polynucleotides encoding the antibodies of the invention or complementary nucleic acids thereof, vectors, host cells, and methods of making and using them.

General Aspects of the Antibodies Described Herein

The anti-CD3 antibodies or antigen-binding fragments described herein include variants having single or multiple amino acid substitutions, deletions, or additions that retain the biological properties (e.g., binding affinity or immune effector activity) of the described anti-CD3 antibodies or antigen-binding fragments. In the context of the present invention the following notations are, unless otherwise indicated, used to describe a mutation; i) substitution of an amino acid in a given position is written as e.g. K409R which means a substitution of a Lysine in position 409 with an Arginine; and ii) for specific variants the specific three or one letter codes are used, including the codes Xaa and X to indicate any amino acid residue. Thus, the substitution of Arginine for Lysine in position 409 is designated as: K409R, or the substitution of any amino acid residue for Lysine in position 409 is designated as K409X. In case of deletion of Lysine in position 409 it is indicated by K409*. The skilled person may produce variants having single or multiple amino acid substitutions, deletions, or additions.

These variants may include: (a) variants in which one or more amino acid residues are substituted with conservative or nonconservative amino acids, (b) variants in which one or more amino acids are added to or deleted from the polypeptide, (c) variants in which one or more amino acids include a substituent group, and (d) variants in which the polypeptide is fused with another peptide or polypeptide such as a fusion partner, a protein tag or other chemical moiety, that may confer useful properties to the polypeptide, such as, for example, an epitope for an antibody, a polyhistidine sequence, a biotin moiety and the like. Antibodies or antigen-binding fragments described herein may include variants in which amino acid residues from one species are substituted for the corresponding residue in another species, either at the conserved or nonconserved positions. In other embodiments, amino acid residues at nonconserved positions are substituted with conservative or nonconservative residues. The techniques for obtaining these variants, including genetic (deletions, mutations, etc.), chemical, and enzymatic techniques, are known to persons having ordinary skill in the art.

The antibodies or antigen-binding fragments described herein may may be an IgM, an IgD, an IgG, an IgA or an IgE isotype. In some embodiments, the antibody isotype is an IgG1, an IgG2, an IgG3, or an IgG4 isotype. In some embodiments, the antibody is an IgG1 isotype. In some embodiments, the antibody is an IgG2 isotype. In some embodiments, the antibody is an IgG3 isotype. In some embodiments, the antibody is an IgG4 isotype. The specificity of the antibody or antigen-binding fragment thereof is in part determined by the amino acid sequence and arrangement, of the CDRs. Therefore, the CDRs of one isotype may be transferred to another isotype without altering antigen specificity. Accordingly, such antibody isotypes are within the scope of the described antibodies or antigen-binding fragments.

The IgG class is divided in four isotypes: IgG1, IgG2, IgG3 and IgG4 in humans. They share more than 95% homology in the amino acid sequences in the CH1, CH2 and CH3 regions but show major differences in the amino acid composition and structure of the hinge region. The Fc region mediates effector functions, such as antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) and complement-dependent cytotoxicity (CDC). In ADCC, the Fc region binds to Fc receptors (FcγRs) on the surface of immune effector cells such as natural killer cells and macrophages, leading to the lysis of the targeted cells. In CDC, the antibodies mediate targeted cell killing by triggering the complement cascade at the cell surface. In ADCP, the antibody mediates elimination of antibody-coated target cells by internalization by phagocytic cells, such as macrophages or dendritic cells. The antibodies described herein include antibodies with the described features of the variable domains in combination with any of the IgG isotypes, including modified versions in which the Fc region has been modified to modulate the various effector functions.

For many applications of therapeutic antibodies, Fc-mediated effector functions are not desired as they may potentially pose a safety risk due to cell depletion. Modifying effector functions can be achieved by engineering the Fc regions to reduce their binding to FcγRs or the complement factors. The binding of IgG to the activating (FcγRI, FcγRIIa, FcγRIIIa and FcγRIIIb) and inhibitory (FcγRIIb) FcγRs or the first component of complement (C1q) depends on residues located in the hinge region and the CH2 domain. Mutations may be introduced in IgG1, IgG2 and IgG4 to reduce or silence Fc-mediated effector functions. The antibodies described herein may include these modifications.

In some embodiments, the anti-CD3, the anti-PSMA, the anti-CD33, and/or the anti-IL1RAP antibodies comprise an engineered Fc region having one or more of the following properties: (a) reduced effector function when compared to the parental Fc; (b) reduced affinity to FcγRI, FcγRIIa, FcγRIIb, FcγRIIIb and/or FcγRIIIa; (c) reduced affinity to FcγRI; (d) reduced affinity to FcγRIIa; (e) reduced affinity to FcγRIIIb; or (f) reduced affinity to FcγRIIIa.

In some embodiments, the anti-CD3, the anti-PSMA, the anti-CD33, and/or the anti-IL1RAP antibodies are, e.g., IgG1, IgG2, IgG3, or IgG4 isotypes. In some embodiments wherein the antibody has an IgG4 isotype, the antibody contains S228P, F234A, and L235A substitutions in its Fc region when compared to the wild-type IgG4. In some embodiments wherein the antibody has an IgG1 isotype, the antibody contains L234A, and L235A substitutions in its Fc region. The antibodies described herein may include these modifications.

In some embodiments, the anti-CD3, the anti-PSMA, the anti-CD33, and/or the anti-IL1RAP antibodies are an IgG4 isotype, optionally comprising a heavy chain substitution S228P.

In some embodiments, the anti-CD3, the anti-PSMA, the anti-CD33, and/or the anti-IL1RAP antibodies are an IgG1 isotype, optionally comprising heavy chain substitutions L234A, G237A, P238S, H268A, A330S and P331S when compared to the wild type IgG1.

In some embodiments, the anti-CD3, the anti-PSMA, the anti-CD33, and/or the anti-IL1RAP antibodies are an IgG2 isotype, optionally comprising heavy chain substitutions L234A, G237A, P238S, H268A, V309L, A330S and P331S when compared to the wild type IgG2.

Wild-type IgG4

(SEQ ID NO: 602)

ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGV

HTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVES

KYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQED

PEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYK

CKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVK

GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEG

NVFSCSVMHEALHNHYTQKSLSLSLGK

Wild-type IgG1

(SEQ ID NO: 601)

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV

HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP

KSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS

HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK

EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTC

LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW

QQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Wild-type IgG2

(SEQ ID NO: 711)

ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGV

HTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVER

KCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP

EVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKC

KVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKG

FYPSDISVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGN

VFSCSVMHEALHNHYTQKSLSLSPGK

In certain embodiments, labelled anti-CD3, anti-PSMA, anti-CD33, and/or anti-IL1RAP antibodies are provided. Exemplary labels or moieties that are detected directly (e.g., fluorescent, chromophoric, electron-dense, chemiluminescent, and radioactive labels) and labels and moieties (e.g., enzymes or ligands) that are detected indirectly (e.g., through enzymatic reaction or molecular interaction). Exemplary labels include radiolabels (e.g., ³²P, ¹⁴C, ¹¹¹I, ¹²⁵I, ³H, ¹³¹I), fluorescent labels (such as DyLight® 649), epitope tags, biotin, chromophore labels, ECL labels, or enzymes. More specifically, the described labels include ruthenium, ¹¹¹In-DOTA, ¹¹¹In-diethylenetriaminepentaacetic acid (DTPA), horseradish peroxidase, alkaline phosphatase and beta-galactosidase, poly-histidine (HIS tag), acridine dyes, cyanine dyes, fluorone dyes, oxazin dyes, phenanthridine dyes, rhodamine dyes, Alexafluor® dyes, and the like.

In addition to the described anti-CD3, anti-PSMA, anti-CD33, and/or anti-IL1RAP antibodies and antigen-binding fragments, also provided are polynucleotide sequences capable of encoding the described antibodies and antigen-binding fragments. Vectors comprising the described polynucleotides are also provided, as are cells expressing the anti-CD3, anti-PSMA, anti-CD33, and/or anti-IL1RAP antibodies or antigen-binding fragments provided herein. Also described are cells capable of expressing the disclosed vectors. These cells may be mammalian cells (such as 293F cells, CHO cells), insect cells (such as Sf7 cells), yeast cells, plant cells, or bacteria cells (such as E. coli). The described antibodies may also be produced by hybridoma cells.

Generation of Monospecific Antibodies

In some embodiments, the anti-CD3, the anti-PSMA, the anti-CD33, the anti-TMEFF2, and/or the anti-IL1RAP antibodies of the invention are human.

In some embodiments, the anti-CD3, the anti-PSMA, the anti-CD33, the anti-TMEFF2, and/or the anti-IL1RAP antibodies of the invention are humanized.

Monospecific antibodies of the invention described herein (e.g. the anti-CD3, the anti-PSMA, the anti-CD33, the anti-TMEFF2, and/or the anti-IL1RAP antibodies) may be generated using various technologies. For example, the hybridoma method of Kohler and Milstein, Nature 256:495, 1975 may be used to generate monoclonal antibodies. In the hybridoma method, a mouse or other host animal, such as a hamster, rat or chicken, is immunized with human, chimpanzee, or macaque PSMA, CD33, IL1RAP, TMEFF2, or CD3 or fragments of PSMA, CD33, IL1RAP, TMEFF2, or CD3, such as the extracellular domain of PSMA, CD33, IL1RAP, TMEFF2, or CD3, followed by fusion of spleen cells from immunized animals with myeloma cells using standard methods to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)). Colonies arising from single immortalized hybridoma cells are screened for production of antibodies with desired properties, such as specificity of binding, cross-reactivity or lack thereof, and affinity for the antigen.

Various host animals may be used to produce the anti-CD3, the anti-PSMA, the anti-CD33, TMEFF2, and/or the anti-IL1RAP antibodies of the invention described herein. For example, Balb/c mice may be used to generate mouse anti-human PSMA antibodies. The antibodies made in Balb/c mice and other non-human animals may be humanized using various technologies to generate more human-like sequences.

Exemplary humanization techniques including selection of human acceptor frameworks are known and include CDR grafting (U.S. Pat. No. 5,225,539), SDR grafting (U.S. Pat. No. 6,818,749), Resurfacing (Padlan, (1991) Mol Immunol 28:489-499), Specificity Determining Residues Resurfacing (U.S. Patent Publ. No. 2010/0261620), human framework adaptation (U.S. Pat. No. 8,748,356) or superhumanization (U.S. Pat. No. 7,709,226). In these methods, CDRs of parental antibodies are transferred onto human frameworks that may be selected based on their overall homology to the parental frameworks, based on similarity in CDR length, or canonical structure identity, or a combination thereof.

Humanized antibodies may be further optimized to improve their selectivity or affinity to a desired antigen by incorporating altered framework support residues to preserve binding affinity (backmutations) by techniques such as those described in Int. Patent Publ. Nos. WO1090/007861 and WO1992/22653, or by introducing variation at any of the CDRs for example to improve affinity of the antibody.

Transgenic animals, such as mice or rat carrying human immunoglobulin (Ig) loci in their genome may be used to generate human antibodies against a target protein, and are described in for example U.S. Pat. No. 6,150,584, Int. Patent Publ. No. WO99/45962, Int. Patent Publ. Nos. WO2002/066630, WO2002/43478, WO2002/043478 and WO1990/04036, Lonberg et al (1994) Nature 368:856-9; Green et al (1994) Nature Genet. 7:13-21; Green & Jakobovits (1998) Exp. Med. 188:483-95; Lonberg and Huszar (1995) Int Rev Immunol 13:65-93; Bruggemann et al., (1991) Eur J Immunol 21:1323-1326; Fishwild et al., (1996) Nat Biotechnol 14:845-851; Mendez et al., (1997) Nat Genet 15:146-156; Green (1999) J Immunol Methods 231:11-23; Yang et al., (1999) Cancer Res 59:1236-1243; Bruggemann and Taussig (1997) Curr Opin Biotechnol 8:455-458. The endogenous immunoglobulin loci in such animal may be disrupted or deleted, and at least one complete or partial human immunoglobulin locus may be inserted into the genome of the animal using homologous or non-homologous recombination, using transchromosomes, or using minigenes. Companies such as Regeneron (http://_www_regeneron_com), Harbour Antibodies (http://_www_harbourantibodies_com), Open Monoclonal Technology, Inc. (OMT) (http://_www_omtinc_net), KyMab (http://_www_kymab_com), Trianni (http://_www.trianni_com) and Ablexis (http://_www_ablexis_com) may be engaged to provide human antibodies directed against a selected antigen using technologies as described above.

Human antibodies may be selected from a phage display library, where the phage is engineered to express human immunoglobulins or portions thereof such as Fabs, single chain antibodies (scFv), or unpaired or paired antibody variable regions (Knappik et al., (2000) J Mol Biol 296:57-86; Krebs et al., (2001) J Immunol Meth 254:67-84; Vaughan et al., (1996) Nature Biotechnology 14:309-314; Sheets et al., (1998) PITAS (USA) 95:6157-6162; Hoogenboom and Winter (1991) J Mol Biol 227:381; Marks et al., (1991) J Mol Biol 222:581). The antibodies of the invention may be isolated for example from phage display library expressing antibody heavy and light chain variable regions as fusion proteins with bacteriophage pIX coat protein as described in Shi et al., (2010) J Mol Biol 397:385-96, and Int. Patent Publ. No. WO09/085462). The libraries may be screened for phage binding to human and/or cyno PSMA, CD33, IL1RAP, TMEFF2, or CD3 and the obtained positive clones may be further characterized, the Fabs isolated from the clone lysates, and expressed as full length IgGs. Such phage display methods for isolating human antibodies are described in for example: U.S. Pat. Nos. 5,223,409, 5,403,484, 5,571,698, 5,427,908, 5,580,717, 5,969,108, 6,172,197, 5,885,793; 6,521,404; 6,544,731; 6,555,313; 6,582,915 and 6,593,081.

Preparation of immunogenic antigens and monoclonal antibody production may be performed using any suitable technique, such as recombinant protein production. The immunogenic antigens may be administered to an animal in the form of purified protein, or protein mixtures including whole cells or cell or tissue extracts, or the antigen may be formed de novo in the animal's body from nucleic acids encoding said antigen or a portion thereof.

Generation and Use of Bispecific and Multispecific CD3 Antibodies

The invention provides bispecific and multispecific antibodies comprising a first domain that specifically binds CD3 and a second domain that specifically binds a second antigen. The second antigen may be a tumor associated antigen (TAA) or an antigen on pathogenic cells.

Exemplary anti-CD3 antibodies that may be used to engineer bispecific and multispecific antibodies comprising a first domain that specifically binds CD3 and a second domain that specifically binds a second antigen include CD3 antibodies comprising the V_H/V_Lsequences and heavy/light chain CDRs shown in Table 7A and 7B, respectively, and the engineered variants thereof described in Table 10 and Table 11, and accompanying text. For example, the CDRs and/or the VH/VL domains of the CD3 antibodies CD3B312, CD3B313, CD3B314, CD3B315, CD3B316, CD3B317, CD3B337, CD3B373, CD3B376, CD3B389, CD3B450, and CD3B467 described herein may be used to generate bispecific and multispecific antibodies comprising a first domain that specifically binds CD3 and a second domain that specifically binds a second antigen.

The herein described anti-CD3 antibody CDRs and/or the VH/VL domains may be incorporated into bispecific antibodies comprising a first domain that specifically binds CD3 and a second domain that specifically binds a second antigen.

The herein described anti-CD3 antibody CDRs and/or the VH/VL domains may be incorporated into bispecific antibodies comprising PSMA binding VH/VL domains described herein and in Table 23. The herein described anti-CD3 antibodies and/or the VH/VL domains may be incorporated into bispecific antibodies comprising IL1RAP binding VH/VL domains described herein and in Table 30. The herein described anti-CD3 antibody CDRs and/or the VH/VL domains may be incorporated into bispecific antibodies comprising CD33 binding VH/VL domains described herein and in Table 38. For example, the VH/VL domains of the PSMA antibodies PSMB119, PSMB120, PSMB121, PSMB122, PSMB123, PSMB87, PSMB126, PSMB127, PSMB128, PSMB129, PSMB130, PSMB120, PSMB121, PSMB122, PSMB123, PSMB127, PSMB128, PSMB130, PSMB344, PSMB345, PSMB346, PSMB347, PSMB349, PSMB358, PSMB359, PSMB360, PSMB361, PSMB362, PSMB363, and PSMB365 described herein may be used to generate bispecific PSMA×CD3 antibodies.

Exemplary TAAs are PSMA, CD33, TMEFF2, and IL1RAP. The exemplary multispecific PSMA×CD3, CD33×CD3, TMEFF2, ×CD3, and IL1RAP×CD3 antibodies provided herein have a first domain that specifically bind CD3 and a second domain that specifically binds PSMA, CD33, TMEFF2, or IL1RAP. Exemplary anti-PSMA antibodies that may be used to engineer bispecific PSMA×CD3 molecules are those described herein, which may comprise heavy and light chain sequences including but not limited to the heavy and light chain sequences listed in Table 23. Exemplary anti-IL1RAP antibodies that may be used to engineer bispecific IL1RAP×CD3 molecules are those described herein, which may comprise heavy and light chain variable region sequences including but not limited to the heavy and light chain variable region sequences provided in Table 35. Exemplary anti-CD33 antibodies that may be used to engineer bispecific CD33×CD3 molecules are those described herein, which may comprise heavy and light chain variable region sequences including but not limited to the heavy and light chain variable region sequences provided in Table 43. Exemplary anti-TMEFF2 antibodies that may be used to engineer bispecific TMEFF2×CD3 molecules are those described herein, which may comprise heavy and light chain variable region sequences including but not limited to the heavy and light chain variable region sequences provided in Tables 59, 66-68.

The generated bispecific antibodies may be tested for their binding to CD3 and/or the second antigen and/or for their desired functional characteristics, such as T-cell mediated killing of cells that express the second antigen.

The bispecific antibodies provided herein include antibodies having a full-length antibody structure.

“Fab-arm” or “half molecule” refers to one heavy chain-light chain pair that specifically binds an antigen.

Full length bispecific antibodies as described herein may be generated for example using Fab arm exchange (or half molecule exchange) between two monospecific bivalent antibodies by introducing substitutions at the heavy chain CH3 interface in each half molecule to favor heterodimer formation of two antibody half molecules having distinct specificity either in vitro in cell-free environment or using co-expression. The Fab arm exchange reaction is the result of a disulfide-bond isomerization reaction and dissociation-association of CH3 domains. The heavy chain disulfide bonds in the hinge regions of the parental monospecific antibodies are reduced. The resulting free cysteines of one of the parental monospecific antibodies form an inter heavy-chain disulfide bond with cysteine residues of a second parental monospecific antibody molecule and simultaneously CH3 domains of the parental antibodies release and reform by dissociation-association. The CH3 domains of the Fab arms may be engineered to favor heterodimerization over homodimerization. The resulting product is a bispecific antibody having two Fab arms or half molecules, which each bind a distinct epitope, i.e. an epitope on CD3 and an epitope on the second antigen.

“Homodimerization” refers to an interaction of two heavy chains having identical CH3 amino acid sequences. “Homodimer” refers to an antibody having two heavy chains with identical CH3 amino acid sequences.

“Heterodimerization” refers to an interaction of two heavy chains having non-identical CH3 amino acid sequences. “Heterodimer” refers to an antibody having two heavy chains with non-identical CH3 amino acid sequences.

In some embodiments, the bispecific antibodies include designs such as the Triomab/Quadroma (Trion Pharma/Fresenius Biotech), Knob-in-Hole (Genentech), CrossMAbs (Roche) and the electrostatically-matched (Chugai, Amgen, NovoNordisk, Oncomed), the LUZ-Y (Genentech), the Strand Exchange Engineered Domain body (SEEDbody)(EMD Serono), the Biclonic (Merus) and the DuoBody® Technology (Genmab A/S).

The Triomab quadroma technology may be used to generate full-length bispecific antibodies, incorporating the VH and VL of the anti-CD3 antibodies of the invention. Triomab technology promotes Fab arm exchange between two parental chimeric antibodies, one parental mAb having IgG2a and the second parental mAb having rat IgG2b constant regions, yielding chimeric bispecific antibodies.

The “knob-in-hole” strategy (see, e.g., Intl. Publ. No. WO 2006/028936) may be used to generate full-length bispecific antibodies. Briefly, selected amino acids forming the interface of the CH3 domains in human IgG can be mutated at positions affecting CH3 domain interactions to promote heterodimer formation. An amino acid with a small side chain (hole) is introduced into a heavy chain of an antibody specifically binding a first antigen and an amino acid with a large side chain (knob) is introduced into a heavy chain of an antibody specifically binding a second antigen. After co-expression of the two antibodies, a heterodimer is formed as a result of the preferential interaction of the heavy chain with a “hole” with the heavy chain with a “knob”. Exemplary CH3 substitution pairs forming a knob and a hole are (expressed as modified position in the first CH3 domain of the first heavy chain/modified position in the second CH3 domain of the second heavy chain): T366Y/F405A, T366W/F405W, F405W/Y407A, T394W/Y407T, T394S/Y407A, T366W/T394S, F405W/T394S and T366W/T366S_L368A_Y407V.

The CrossMAb technology may be used to generate full-length bispecific antibodies. CrossMAbs, in addition to utilizing the “knob-in-hole” strategy to promoter Fab arm exchange, have in one of the half arms the CH1 and the CL domains exchanged to ensure correct light chain pairing of the resulting bispecific antibody (see e.g. U.S. Pat. No. 8,242,247).

Other cross-over strategies may be used to generate full length bispecific antibodies by exchanging variable or constant, or both domains between the heavy chain and the light chain or within the heavy chain in the bispecific antibodies, either in one or both arms. These exchanges include for example VH-CH1 with VL-CL, VH with VL, CH3 with CL and CH3 with CH1 as described in Int. Patent Publ. Nos. WO2009/080254, WO2009/080251, WO2009/018386 and WO2009/080252.

Other strategies such as promoting heavy chain heterodimerization using electrostatic interactions by substituting positively charged residues at one CH3 surface and negatively charged residues at a second CH3 surface may be used, as described in US Patent Publ. No. US2010/0015133; US Patent Publ. No. US2009/0182127; US Patent Publ. No. US2010/028637 or US Patent Publ. No. US2011/0123532. In other strategies, heterodimerization may be promoted by following substitutions (expressed as modified position in the first CH3 domain of the first heavy chain/modified position in the second CH3 domain of the second heavy chain): L351Y_F405A_Y407V/T394W, T366I_K392M_T394W/F405A_Y407V, T366L_K392M_T394W/F405A_Y407V, L351Y_Y407A/T366A_K409F, L351Y_Y407A/T366V_K409F, Y407A/T366A_K409F, or T350V_L351Y_F405A_Y407V/T350V_T366L_K392L_T394W as described in U.S. Patent Publ. No. US2012/0149876 or U.S. Patent Publ. No. US2013/0195849.

LUZ-Y technology may be utilized to generate bispecific antibodies. In this technology, a leucine zipper is added into the C terminus of the CH3 domains to drive the heterodimer assembly from parental mAbs that is removed post-purification as described in Wranik et al., (2012) J Biol Chem 287(52): 42221-9.

SEEDbody technology may be utilized to generate bispecific antibodies. SEEDbodies have, in their constant domains, select IgG residues substituted with IgA residues to promote heterodimerization as described in U.S. Patent No. US20070287170.

The bispecific antibodies described herein may be generated in vitro in a cell-free environment by introducing asymmetrical mutations in the CH3 regions of two monospecific homodimeric antibodies and forming the bispecific heterodimeric antibody from two parent monospecific homodimeric antibodies in reducing conditions to allow disulfide bond isomerization according to methods described in Int. Patent Publ. No. WO2011/131746). In the methods, the first monospecific bivalent antibody (i.e., the antibody that specifically binds the second antigen; e.g., anti-PSMA, anti-CD33, anti-TMEFF2 or anti-IL1RAP antibody) and the second monospecific bivalent antibody (i.e., anti-CD3 antibody) are engineered to have certain substitutions at the CH3 domain that promoter heterodimer stability; the antibodies are incubated together under reducing conditions sufficient to allow the cysteines in the hinge region to undergo disulfide bond isomerization; thereby generating the bispecific antibody by Fab arm exchange. The incubation conditions are restored to non-reducing. Exemplary reducing agents that may be used are 2-mercaptoethylamine (2-MEA), dithiothreitol (DTT), dithioerythritol (DTE), glutathione, tris(2-carboxyethyl)phosphine (TCEP), L-cysteine and beta-mercaptoethanol. For example, incubation for at least 90 min at a temperature of at least 20° C. in the presence of at least 25 mM 2-MEA or in the presence of at least 0.5 mM dithiothreitol at a pH of from 5-8, for example at pH of 7.0 or at pH of 7.4 may be used.

In some embodiments described herein, the bispecific antibody comprising a first domain that specifically binds CD3 and a second domain that specifically binds a second antigen comprises at least one substitution in an antibody CH3 constant domain.

In some embodiments described herein, the at least one substitution in the antibody CH3 constant domain is K409R, F405L or F405L and R409K substitution, wherein residue numbering is according to the EU Index.

Antibody domains and numbering are well known. “Asymmetrical” refers to non-identical substitutions in the two CH3 domains in two separate heavy chains in an antibody. An IgG1 CH3 region typically consists of residues 341-446 on IgG1 (residue numbering according to the EU index).

In some embodiments described herein, the bispecific antibody is an IgG1 isotype and comprises a F405L substitution in an antibody first heavy chain (HC1) and a K409R substitution in an antibody second heavy chain (HC2) when compared to the wild-type IgG1.

In some embodiments described herein, the bispecific antibody is an IgG1 isotype and comprises a K409R substitution in an antibody first heavy chain (HC1) and a F405L substitution in an antibody second heavy chain (HC2) when compared to the wild-type IgG1.

In some embodiments described herein, the bispecific antibody is an IgG4 isotype and comprises a S228P substitution in the HC1 and S228P, F405L and R409K substitutions in the HC2 when compared to the wild-type IgG4.

In some embodiments described herein, the bispecific antibody is an IgG4 isotype and comprises S228P, F405L and R409K substitutions in the HC1 and a S228P substitution in the HC2 when compared to the wild-type IgG4.

In some embodiments described herein, the bispecific antibody is an IgG4 isotype and comprises S228P, F234A and L235A substitutions in the HC1 and S228P, F234A, L235A, F405L and R409K substitutions in the HC2 when compared to the wild-type IgG4.

In some embodiments described herein, the bispecific antibody is an IgG4 isotype and comprises S228P, F234A, L235A, F405L and R409K substitutions in the HC1 and a S228P, F234A and L235A substitutions in the HC2 when compared to the wild-type IgG4.

In some embodiments described herein, the bispecific antibody of the invention comprises at least one, two, three, four, five, six, seven or eight asymmetrical substitutions in the HC1 and the HC2 at residue positions 350, 366, 368, 370, 399, 405, 407 or 409, when residue numbering is according to the EU index.

In some embodiments described herein, the bispecific antibody of the invention comprises at least one, two, three or four asymmetrical substitutions in the HC1 and the HC2 at residue positions 350, 370, 405 or 409, when residue numbering is according to the EU index.

In some embodiments described herein, the bispecific antibody of the invention comprises at least one asymmetrical substitution in the HC1 and the HC2 at residue positions 405 or 409, when residue numbering is according to the EU index.

Substitutions are typically made at the DNA level to a molecule such as the constant domain of the antibody using standard methods.

The antibodies of the invention may be engineered into various well-known antibody forms.

In some embodiments, the bispecific antibody of the present invention is a cross-body.

In some embodiments, the bispecific antibodies of the invention include recombinant IgG-like dual targeting molecules, wherein the two sides of the molecule each contain the Fab fragment or part of the Fab fragment of at least two different antibodies; IgG fusion molecules, wherein full length IgG antibodies are fused to an extra Fab fragment or parts of Fab fragment; Fc fusion molecules, wherein single chain Fv molecules or stabilized diabodies are fused to heavy-chain constant-domains, Fc-regions or parts thereof; Fab fusion molecules, wherein different Fab-fragments are fused together; ScFv- and diabody-based and heavy chain antibodies (e.g., domain antibodies, nanobodies) wherein different single chain Fv molecules or different diabodies or different heavy-chain antibodies (e.g. domain antibodies, nanobodies) are fused to each other or to another protein or carrier molecule.

In some embodiments, recombinant IgG-like dual targeting molecules include Dual Targeting (DT)-Ig (GSK/Domantis), Two-in-one Antibody (Genentech) and mAb2 (F-Star).

In some embodiments, IgG fusion molecules include Dual Variable Domain (DVD)-Ig (Abbott), Ts2Ab (MedImmune/AZ) and BsAb (Zymogenetics), HERCULES (Biogen Idec) and TvAb (Roche).

In some embodiments, Fc fusion molecules include to ScFv/Fc Fusions (Academic Institution), SCORPION (Emergent BioSolutions/Trubion, Zymogenetics/BMS) and Dual Affinity Retargeting Technology (Fc-DART) (MacroGenics).

In some embodiments, Fab fusion bispecific antibodies include F(ab)2 (Medarex/AMGEN), Dual-Action or Bis-Fab (Genentech), Dock-and-Lock (DNL) (ImmunoMedics), Bivalent Bispecific (Biotecnol) and Fab-Fv (UCB-Celltech). ScFv-, diabody-based and domain antibodies include Bispecific T Cell Engager (BITE) (Micromet), Tandem Diabody (Tandab) (Affimed), Dual Affinity Retargeting Technology (DART) (MacroGenics), Single-chain Diabody (Academic), TCR-like Antibodies (AIT, ReceptorLogics), Human Serum Albumin ScFv Fusion (Merrimack) and COMBODY (Epigen Biotech), dual targeting nanobodies (Ablynx), dual targeting heavy chain only domain antibodies. Various formats of bispecific antibodies have been described, for example in Chames and Baty (2009) Curr Opin Drug Disc Dev 12: 276 and in Nunez-Prado et al., (2015) Drug Discovery Today 20(5):588-594.

Table 2 summarizes exemplary monoclonal antibodies described herein that can be used to generate the bispecific antibodies of the invention.

TABLE 2

Exemplary monoclonal antibodies that can be used to

generate the bispecific antibodies of the invention

First Domain
Second Domain

anti-CD3
anti-PSMA
anti-CD33
anti-IL1RAP

CD3B312
PSMB87
C33B760
IAPB3

CD3B313
PSMB119
C33B777
IAPB9

CD3B314
PSMB120
C33B778
IAPB17

CD3B315
PSMB121
C33B782
IAPB23

CD3B316
PSMB122
C33B792
IAPB25

CD3B317
PSMB123
C33B799
IAPB29

CD3B337
PSMB124
C33B806
IAPB38

CD3B373
PSMB126
C33B830
IAPB47

CD3B376
PSMB127
C33B836
IAPB55

CD3B389
PSMB129
C33B903
IAPB57

CD3B450
PSMB130
C33B904
IAPB61

CD3B467
PSMB344
C33B905
IAPB62

PSMB345
C33B907
IAPB63

PSMB346
C33B908
IAPB64

PSMB347

IAPB65

PSMB349

PSMB358

PSMB359

PSMB360

PSMB361

PSMB362

PSMB363

PSMB365

Exemplary anti-TMEFF2 antibodies that may be used to engineer bispecific TMEFF2×CD3 molecules are those described herein, which may comprise heavy and light chain variable region sequences including but not limited to the heavy and light chain variable region sequences provided in Tables 59, 66-68.

In some embodiments, the anti-CD3 antibody is a multispecific antibody, for example a bispecific antibody comprising a first domain that specifically binds CD3 and a second domain that specifically binds a second antigen. In some embodiments, the second, or target, antigen is a cell surface antigen that is expressed on a target cell other than an immune effector cell. In some embodiments, the second antigen is a TAA. Exemplary TAAs are PSMA, CD33, TMEFF2, and IL1RAP.

The invention provides a bispecific antibody comprising a first domain that specifically binds CD3 and a second domain that specifically binds a second antigen.

In some embodiments, the first domain comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2, and the LCDR3 of SEQ ID NOs:662, 663, 664, 671, 673, and 690, respectively. In some embodiments, the first domain comprises the VH and VL of SEQ ID NOs: 652 and 661, respectively. In some embodiments, the first domain comprises the HC and LC of SEQ ID NOs: 640 and 676, respectively. In some embodiments, the first domain comprises the VH and VL of SEQ ID NOs: 657 and 678, respectively. In some embodiments, the first domain comprises the HC and LC of SEQ ID NOs: 675 and 677, respectively.

In some embodiments, the first domain comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2, and the LCDR3 of SEQ ID NOs:662, 663, 664, 773, 673, and 690, respectively. In some embodiments, the first domain comprises the VH and VL of SEQ ID NOs:657 and 678, respectively. In some embodiments, the first domain comprises the HC and LC of SEQ ID NOs:675 and 677, respectively.

The invention also provides the bispecific antibody comprising a first domain that specifically binds CD3 and a second domain that specifically binds a second antigen of the invention for use in therapy.

A further aspect of the invention is a method of treating a cell proliferative disorder or an autoimmune disorder in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the anti-CD3 antibody of the invention. In some embodiments, the anti-CD3 antibody or the bispecific antibody comprising a first domain that specifically binds CD3 and a second domain that specifically binds a second antigen of the invention is administered to the subject in a dosage of about 0.01 mg/kg to about 10 mg/kg. In some embodiments, the anti-CD3 antibody or the bispecific antibody comprising a first domain that specifically binds CD3 and a second domain that specifically binds a second antigen of the invention is administered to the subject in a dosage of about 0.1 mg/kg to about 10 mg/kg. In some embodiments, the anti-CD3 antibody or the bispecific antibody comprising a first domain that specifically binds CD3 and a second domain that specifically binds a second antigen of the invention is administered to the subject in a dosage of about 1 mg/kg. In some embodiments, the anti-CD3 antibody or the bispecific antibody comprising a first domain that specifically binds CD3 and a second domain that specifically binds a second antigen of the invention is administered subcutaneously, intravenously, intramuscularly, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally. In some embodiments, the anti-CD3 antibody or the bispecific antibody comprising a first domain that specifically binds CD3 and a second domain that specifically binds a second antigen of the invention is administered subcutaneously. In some embodiments, the anti-CD3 antibody or the bispecific antibody comprising a first domain that specifically binds CD3 and a second domain that specifically binds a second antigen of the invention is administered intravenously.

In any of the preceding uses or methods, the cell proliferative disorder is cancer. In some embodiments, the cancer is selected from the group consisting of esophageal cancer, stomach cancer, small intestine cancer, large intestine cancer, colorectal cancer, breast cancer, non-small cell lung cancer, non-Hodgkin's lymphoma (NHL), B cell lymphoma, B cell leukemia, multiple myeloma, renal cancer, prostate cancer, liver cancer, head and neck cancer, melanoma, ovarian cancer, mesothelioma, glioblastoma, germinal-center B-cell-like (GCB) DLBCL, activated B-cell-like (ABC) DLBCL, follicular lymphoma (FL), mantle cell lymphoma (MCL), acute myeloid leukemia (AML), chronic lymphoid leukemia (CLL), marginal zone lymphoma (MZL), small lymphocytic leukemia (SLL), lymphoplasmacytic lymphoma (LL), Waldenstrom 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 variant, Waldenstrom macroglobulinemia, Heavy chain diseases, Plasma cell myeloma, Solitary plasmacytoma of bone, Extraosseous plasmacytoma, Extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma), Nodal marginal zone lymphoma, Pediatric nodal marginal zone lymphoma, Pediatric follicular lymphoma, Primary cutaneous follicle centre 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 granulomatosis, Primary mediastinal (thymic) large B-cell lymphoma. Intravascular large B-cell lymphoma, ALK-positive large B-cell lymphoma, Plasmablastic lymphoma, Large B-cell lymphoma arising in HHV8-associated multicentric Castleman disease, Primary effusion lymphoma: B-cell lymphoma, unclassifiable, with features intermediate between diffuse large B-cell lymphoma and Burkitt lymphoma, and B-cell lymphoma, unclassifiable, with features intermediate between diffuse large B-cell lymphoma, classical Hodgkin lymphoma and light chain amyloidosis.

In some embodiments, the cancer is esophageal cancer. In some embodiments, the cancer is an adenocarcinoma, for example, a metastatic adenocarcinoma (e.g., a colorectal adenocarcinoma, a gastric adenocarcinoma, or a pancreatic adenocarcinoma).

In any of the preceding uses or methods, the autoimmune disorder can be selected from the group consisting of rheumatoid arthritis, juvenile rheumatoid arthritis, systemic lupus erythematosus (SLE), Wegener's disease, inflammatory bowel disease, idiopathic thrombocytopenic purpura (ITP), thrombotic thrombocytopenic purpura (TTP), autoimmune thrombocytopenia, multiple sclerosis, psoriasis, IgA nephropathy, IgM polyneuropathies, myasthenia gravis, vasculitis, diabetes mellitus, Reynaud's syndrome, Sjorgen's syndrome, glomerulonephritis, Neuromyelitis Optica (NMO), and IgG neuropathy.

In another aspect, the invention features a kit comprising: (a) a composition comprising any one of the preceding anti-CD3 antibodies or the bispecific antibodies comprising a first domain that specifically binds CD3 and a second domain that specifically binds a second antigen of the invention and (b) a package insert comprising instructions for administering the composition to a subject to treat or delay progression of a cell proliferative disorder. The term “package insert” is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, combination therapy, contraindications and/or warnings concerning the use of such therapeutic products.

In any of the preceding uses or methods, the subject can be a human.

Polynucleotides, Vectors, and Host Cells

Also disclosed are isolated polynucleotides that encode the anti-CD3 antibodies of the invention or the bispecific antibodies comprising a first domain that specifically binds CD3 and a second domain that specifically binds a second antigen of the invention. The isolated polynucleotides capable of encoding the variable domains provided herein may be included on the same, or different, vectors to produce antibodies or antigen-binding fragments of the invention.

In some embodiments, the polynucleotides of the invention include a polynucleotide encoding a leader sequence. Any leader sequence known in the art may be employed. The polynucleotide encoding the leader sequence may include a restriction endonuclease cleavage site or a translation initiation site.

Also provided are vectors comprising the polynucleotides of the invention. The vectors can be expression vectors. The expression vector may contain one or more additional sequences such as but not limited to regulatory sequences (e.g., promoter, enhancer), a selection marker, and a polyadenylation signal. Vectors for transforming a wide variety of host cells are well known and include, but are not limited to, plasmids, phagemids, cosmids, baculoviruses, bacmids, bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), as well as other bacterial, yeast and viral vectors.

Recombinant expression vectors within the scope of the description include synthetic, or cDNA-derived nucleic acid fragments that encode at least one recombinant protein which may be operably linked to suitable regulatory elements. Such regulatory elements may include a transcriptional promoter, sequences encoding suitable mRNA ribosomal binding sites, and sequences that control the termination of transcription and translation. Expression vectors, especially mammalian expression vectors, may also include one or more nontranscribed elements such as an origin of replication, a suitable promoter and enhancer linked to the gene to be expressed, other 5′ or 3′ flanking nontranscribed sequences, 5′ or 3′ nontranslated sequences (such as necessary ribosome binding sites), a polyadenylation site, splice donor and acceptor sites, or transcriptional termination sequences. An origin of replication that confers the ability to replicate in a host may also be incorporated.

The transcriptional and translational control sequences in expression vectors to be used in transforming vertebrate cells may be provided by viral sources. Exemplary vectors may be constructed as described by Okayama and Berg, 3 Mol. Cell. Biol. 280 (1983).

In some embodiments, the antibody- or antigen-binding fragment-coding sequence is placed under control of a powerful constitutive promoter, such as the promoters for the following genes: hypoxanthine phosphoribosyl transferase (HPRT), adenosine deaminase, pyruvate kinase, beta-actin, human myosin, human hemoglobin, human muscle creatine, and others. In addition, many viral promoters function constitutively in eukaryotic cells and are suitable for use with the described embodiments. Such viral promoters include without limitation, Cytomegalovirus (CMV) immediate early promoter, the early and late promoters of SV40, the Mouse Mammary Tumor Virus (MMTV) promoter, the long terminal repeats (LTRs) of Maloney leukemia virus, Human Immunodeficiency Virus (HIV), Epstein Barr Virus (EBV), Rous Sarcoma Virus (RSV), and other retroviruses, and the thymidine kinase promoter of Herpes Simplex Virus. In one embodiment, the PSMA-specific antibody or antigen-binding fragment thereof coding sequence is placed under control of an inducible promoter such as the metallothionein promoter, tetracycline-inducible promoter, doxycycline-inducible promoter, promoters that contain one or more interferon-stimulated response elements (ISRE) such as protein kinase R 2′,5′-oligoadenylate synthetases, Mx genes, ADAR1, and the like.

Vectors described herein may contain one or more Internal Ribosome Entry Site(s) (IRES). Inclusion of an IRES sequence into fusion vectors may be beneficial for enhancing expression of some proteins. In some embodiments the vector system will include one or more polyadenylation sites (e.g., SV40), which may be upstream or downstream of any of the aforementioned nucleic acid sequences. Vector components may be contiguously linked, or arranged in a manner that provides optimal spacing for expressing the gene products (i.e., by the introduction of “spacer” nucleotides between the ORFs), or positioned in another way. Regulatory elements, such as the IRES motif, may also be arranged to provide optimal spacing for expression.

The vectors may comprise selection markers, which are well known in the art. Selection markers include positive and negative selection markers, for example, antibiotic resistance genes (e.g., neomycin resistance gene, a hygromycin resistance gene, a kanamycin resistance gene, a tetracycline resistance gene, a penicillin resistance gene), glutamate synthase genes, HSV-TK, HSV-TK derivatives for ganciclovir selection, or bacterial purine nucleoside phosphorylase gene for 6-methylpurine selection (Gadi et al., 7 Gene Ther. 1738-1743 (2000)). A nucleic acid sequence encoding a selection marker or the cloning site may be upstream or downstream of a nucleic acid sequence encoding a polypeptide of interest or cloning site.

The vectors described herein may be used to transform various cells with the genes encoding the described antibodies or antigen-binding fragments. For example, the vectors may be used to generate anti-CD3, anti-PSMA, anti-CD33, anti-TMEFF2, or anti-IL1RAP antibodies or antigen-binding fragment-producing cells. Thus, the invention also provides a host cell comprising the vectors of the invention.

Numerous techniques are known in the art for the introduction of foreign genes into cells and may be used to construct the recombinant cells for purposes of carrying out the described methods, in accordance with the various embodiments described and exemplified herein. The technique used should provide for the stable transfer of the heterologous gene sequence to the host cell, such that the heterologous gene sequence is heritable and expressible by the cell progeny, and so that the necessary development and physiological functions of the recipient cells are not disrupted. Techniques which may be used include but are not limited to chromosome transfer (e.g., cell fusion, chromosome mediated gene transfer, micro cell mediated gene transfer), physical methods (e.g., transfection, spheroplast fusion, microinjection, electroporation, liposome carrier), viral vector transfer (e.g., recombinant DNA viruses, recombinant RNA viruses) and the like (described in Cline, 29 Pharmac. Ther. 69-92 (1985)). Calcium phosphate precipitation and polyethylene glycol (PEG)-induced fusion of bacterial protoplasts with mammalian cells may also be used to transform cells.

Cells suitable for use in the expression of the antibodies or antigen-binding fragments described herein are preferably eukaryotic cells, more preferably cells of plant, rodent, or human origin, for example but not limited to NSO, CHO, CHOK1, perC.6, Tk-ts13, BHK, HEK293 cells, COS-7, T98G, CV-1/EBNA, L cells, C127, 3T3, HeLa, NS1, Sp2/0 myeloma cells, and BHK cell lines, among others. In addition, expression of antibodies may be accomplished using hybridoma cells. Methods for producing hybridomas are well established in the art.

Cells transformed with expression vectors of the invention may be selected or screened for recombinant expression of the antibodies or antigen-binding fragments of the invention. Recombinant-positive cells are expanded and screened for subclones exhibiting a desired phenotype, such as high level expression, enhanced growth properties, or the ability to yield proteins with desired biochemical characteristics, for example, due to protein modification or altered post-translational modifications. These phenotypes may be due to inherent properties of a given subclone or to mutation. Mutations may be effected through the use of chemicals, UV-wavelength light, radiation, viruses, insertional mutagens, inhibition of DNA mismatch repair, or a combination of such methods.

Pharmaceutical Compositions/Administration

The invention also provides for pharmaceutical compositions comprising the antibodies of the invention and a pharmaceutically acceptable carrier. For therapeutic use, the antibodies of the invention may be prepared as pharmaceutical compositions containing an effective amount of the antibody as an active ingredient in a pharmaceutically acceptable carrier. “Carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the antibody of the invention is administered. Such vehicles may be liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. For example, 0.4% saline and 0.3% glycine may be used. These solutions are sterile and generally free of particulate matter. They may be sterilized by conventional, well-known sterilization techniques (e.g., filtration). The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, stabilizing, thickening, lubricating and coloring agents, etc. The concentration of the antibodies of the invention in such pharmaceutical formulation may vary, from less than about 0.5%, usually to at least about 1% to as much as 15 or 20% by weight and may be selected primarily based on required dose, fluid volumes, viscosities, etc., according to the particular mode of administration selected. Suitable vehicles and formulations, inclusive of other human proteins, e.g., human serum albumin, are described, for example, in e.g. Remington: The Science and Practice of Pharmacy, 21st Edition, Troy, D. B. ed., Lipincott Williams and Wilkins, Philadelphia, P A 2006, Part 5, Pharmaceutical Manufacturing pp 691-1092, See especially pp. 958-989.

The mode of administration for therapeutic use of the antibodies of the invention may be any suitable route that delivers the antibody to the host, such as parenteral administration, e.g., intradermal, intramuscular, intraperitoneal, intravenous or subcutaneous, pulmonary, transmucosal (oral, intranasal, intravaginal, rectal), using a formulation in a tablet, capsule, solution, powder, gel, particle; and contained in a syringe, an implanted device, osmotic pump, cartridge, micropump; or other means appreciated by the skilled artisan, as well known in the art. Site specific administration may be achieved by for example intratumoral, intrarticular, intrabronchial, intraabdominal, intracapsular, intracartilaginous, intracavitary, intracelial, intracerebellar, intracerebroventricular, intracolic, intracervical, intragastric, intrahepatic, intracardial, intraosteal, intrapelvic, intrapericardiac, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intravascular, intravesical, intralesional, vaginal, rectal, buccal, sublingual, intranasal, or transdermal delivery.

The antibodies of the invention may be administered to a subject by any suitable route, for example parentally by intravenous (i.v.) infusion or bolus injection, intramuscularly or subcutaneously or intraperitoneally. i.v. infusion may be given over for example 15, 30, 60, 90, 120, 180, or 240 minutes, or from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 hours.

The dose given to a subject is sufficient to alleviate or at least partially arrest the disease being treated (“therapeutically effective amount”) and may be sometimes 0.005 mg to about 100 mg/kg, e.g. about 0.05 mg to about 30 mg/kg or about 5 mg to about 25 mg/kg, or about 4 mg/kg, about 8 mg/kg, about 16 mg/kg or about 24 mg/kg, or for example about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mg/kg, but may even higher, for example about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, 50, 60, 70, 80, 90 or 100 mg/kg.

A fixed unit dose may also be given, for example, 50, 100, 200, 500 or 1000 mg, or the dose may be based on the patient's surface area, e.g., 500, 400, 300, 250, 200, or 100 mg/m2. Usually between 1 and 8 doses, (e.g., 1, 2, 3, 4, 5, 6, 7 or 8) may be administered to treat the patient, but 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more doses may be given.

The administration of the antibodies of the invention may be repeated after one day, two days, three days, four days, five days, six days, one week, two weeks, three weeks, one month, five weeks, six weeks, seven weeks, two months, three months, four months, five months, six months or longer. Repeated courses of treatment are also possible, as is chronic administration. The repeated administration may be at the same dose or at a different dose. For example, the antibodies of the invention described herein may be administered at 8 mg/kg or at 16 mg/kg at weekly interval for 8 weeks, followed by administration at 8 mg/kg or at 16 mg/kg every two weeks for an additional 16 weeks, followed by administration at 8 mg/kg or at 16 mg/kg every four weeks by intravenous infusion.

For example, the antibodies in the methods described herein, may be provided as a daily dosage in an amount of about 0.1-100 mg/kg, such as 0.5, 0.9, 1.0, 1.1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45, 50, 60, 70, 80, 90 or 100 mg/kg, per day, on at least one of day 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40, or alternatively, at least one of week 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 after initiation of treatment, or any combination thereof, using single or divided doses of every 24, 12, 8, 6, 4, or 2 hours, or any combination thereof.

The antibodies in the methods described herein may also be administered prophylactically in order to reduce the risk of developing cancer, delay the onset of the occurrence of an event in cancer progression, and/or reduce the risk of recurrence when a cancer is in remission.

The antibodies provided herein may be lyophilized for storage and reconstituted in a suitable carrier prior to use. This technique has been shown to be effective with conventional protein preparations and well-known lyophilization and reconstitution techniques can be employed.

Methods of Detecting CD3, PSMA, CD33, IL1RAP, TMEFF2 or Target Antigen and CD3

Provided herein are methods of detecting CD3, PSMA, CD33, TMEFF2, or IL1RAP in a sample, comprising obtaining the sample, contacting the sample with the anti-CD3, the anti-PSMA, the anti-CD33, anti-TMEFF2, or the anti-IL1RAP antibody of the invention and detecting the antibody bound to CD3, PSMA, CD33, TMEFF2, or IL1RAP in the sample.

Further provided are methods of detecting CD3 and the second antigen (for example PSMA, CD33, TMEFF2 or IL1RAP) in a sample, comprising obtaining the sample, contacting the sample with the bispecific antibody comprising a first domain that specifically binds CD3 and a second domain that specifically binds a second antigen, and detecting the antibody bound to CD3 and the second antigen in the sample.

In some embodiments described herein, the sample may be derived from urine, blood, serum, plasma, saliva, ascites, circulating cells, circulating tumor cells, cells that are not tissue associated (i.e., free cells), tissues (e.g., surgically resected tumor tissue, biopsies, including fine needle aspiration), histological preparations, and the like.

The antibodies of the invention may be detected using known methods. Exemplary methods include direct labeling of the antibodies using fluorescent or chemiluminescent labels, or radiolabels, or attaching to the antibodies of the invention a moiety which is readily detectable, such as biotin, enzymes or epitope tags. Exemplary labels and moieties are ruthenium, ¹¹¹In-DOTA, ¹¹¹In-diethylenetriaminepentaacetic acid (DTPA), horseradish peroxidase, alkaline phosphatase and beta-galactosidase, poly-histidine (HIS tag), acridine dyes, cyanine dyes, fluorone dyes, oxazin dyes, phenanthridine dyes, rhodamine dyes and Alexafluor® dyes.

The antibodies provided herein may be used in a variety of assays to detect CD3, PSMA, CD33, IL1RAP, TMEFF2 or CD3 and the second antigenin the sample. Exemplary assays are western blot analysis, radioimmunoassay, surface plasmon resonance, immunoprecipitation, equilibrium dialysis, immunodiffusion, electrochemiluminescence (ECL) immunoassay, immunohistochemistry, fluorescence-activated cell sorting (FACS) or ELISA assay.

Antibody Kits

The invention also provides a kit comprising one or more of the anti-CD3, the anti-PSMA, the anti-CD33, anti-TMEFF2 or the anti-IL1RAP antibodies or the bispecific antibodies comprising a first domain that specifically binds CD3 and a second domain that specifically binds a second antigen of the invention or antigen-binding fragments thereof. The described kits may be used to carry out the methods of using the anti-CD3, the anti-PSMA, the anti-CD33, anti-TMEFF2 or the anti-IL1RAP antibodies or the bispecific antibodies comprising a first domain that specifically binds CD3 and a second domain that specifically binds a second antigen of the invention or other methods known to those skilled in the art. In some embodiments the described kits may include the antibodies or antigen-binding fragments described herein and reagents for use in detecting the presence of CD3 or the second antigen such as PSMA, CD33, IL1RAP, TMEFF2 in a biological sample Accordingly, the described kits may include one or more of the antibodies, or an antigen-binding fragment(s) thereof, described herein and a vessel for containing the antibody or fragment when not in use, instructions for use of the antibody or fragment, the antibody or fragment affixed to a solid support, and/or detectably labeled forms of the antibody or fragment, as described herein.

The invention also provides a kit comprising the herein described antibody that specifically binds PSMA. The invention also provides a kit comprising the herein described antibody specifically binding CD33. The invention also provides a kit comprising the herein described antibody specifically binding IL1RAP. The invention also provides a kit comprising the herein described antibody specifically binding TMEFF2.

The kit may be used for therapeutic uses and as diagnostic kits.

The kit may be used to detect the presence of CD3, PSMA, CD33, IL1RAP, TMEFF2 and/or the second antigen in a biological sample.

In some embodiments, the kit comprises the antibody of the invention described herein and reagents for detecting the antibody. The kit can include one or more other elements including: instructions for use; other reagents, e.g., a label, a therapeutic agent, or an agent useful for chelating, or otherwise coupling, an antibody to a label or therapeutic agent, or a radioprotective composition; devices or other materials for preparing the antibody for administration; pharmaceutically acceptable carriers; and devices or other materials for administration to a subject.

In some embodiments, the kit comprises the antibody of the invention in a container and instructions for use of the kit.

In some embodiments, the antibody in the kit is labeled.

CD3-Specific Antibodies

Described herein are isolated anti-CD3 antibodies. The anti-CD3 antibodies of the invention bind human CD3 and optionally cynomolgus CD3. In some embodiments, the anti-CD3 antibodies of the invention and fragments thereof bind to human and cynomolgus CD3 with affinities within 5-fold of each other. In other words, the difference in antibody binding is less than a multiple of 5. In this case, the anti-CD3 antibody of the invention can be used for both preclinical evaluation of safety, activity and/or pharmacokinetic profile of CD3 in primates and as a drug in humans. Put in other words, the same CD3-specific molecule can be used in preclinical animal studies as well as in clinical studies in humans. This human/cyno cross-reactivity leads to highly comparable results and a much-increased predictive power of the animal studies compared to species-specific surrogate molecules. In some embodiments, the anti-CD3 antibodies and fragments thereof of the invention bind an epitope formed by the CD3e/d subunits. The CD3-specific antibodies may be human, humanized, or chimeric. Also exemplified herein are human antibodies generated in OmniRat (Open Monoclonal Technologies (“OMT”), Palo Alto, California, USA, omniab.com).

In some embodiments described herein, the anti-CD3 antibody or fragment thereof has one, two, three, four or five of the following properties:

- a) binds human and Macaca fascicularis CD3+T lymphocytes with a calculated EC50 of nM or less and binds Macaca fascicularis CD3-expressing HEK cells with a calculated EC50 of 40 nM or less, wherein the difference in calculated EC50 between binding CD3+T lymphocytes and binding Macaca fascicularis CD3-expressing HEK cells is less than 5-fold, and wherein the calculated EC50 is measured in a whole cell binding assay at 0° C. using flow cytometry;
- b) binds recombinant CD3d from human (SEQ ID NO:691), or binds recombinant CD3e from human (SEQ ID NO:636), or binds recombinant CD3e from Macaca fascicularis (SEQ ID NO:693) with an equilibrium dissociation constant (K_D) of 12 nM or less, wherein the K_Dis measured using Proteon surface plasmon resonance assay ProteOn XPR36 system at +25° C.;
- c) displays no methionine or tryptophan oxidation, or displays no asparagine deamidation, or displays no asparagine isomerization as detected by peptide mapping analysis;
- d) binds residues 1-6 of CD3e as determined by X-ray crystallography; or
- e) activates T cells or induces CD69 expression to a similar degree as cOKT3 or SP34-2 as determined by fluorescence-activated cell sorting assay.

The anti-CD3 antibodies and fragments thereof of the invention have an in vitro binding affinity (K_d) to human T cells expressing human CD3 that is from about 5 nM to about 1000 nM, preferably from about 5 nM to about 50 nM, from about 50 nM to about 100 nM, from about 100 nM to about 200 nM, from about 200 nM to about 300 nM, from about 300 nM to about 400 nM, from about 400 nM to about 500 nM, from about 500 nM to about 600 nM, from about 600 nM to about 700 nM, from about 700 nM to about 800 nM, from about 800 nM to about 900 nM, and from about 900 nM to about 1000 nM, more preferably from about 5 nM to about 300 nM, as determined by flow cytometry.

In some aspects, the anti-CD3 antibodies and fragments thereof of the invention are bivalent antibodies having an in vitro binding affinity (K_d) to human T cells expressing human CD3 that is from about 5 nM to about 1000 nM preferably from about 5 nM to about 50 nM, from about 50 nM to about 100 nM, from about 100 nM to about 200 nM, from about 200 nM to about 300 nM, from about 300 nM to about 400 nM, from about 400 nM to about 500 nM, from about 500 nM to about 600 nM, from about 600 nM to about 700 nM, from about 700 nM to about 800 nM, from about 800 nM to about 900 nM, and from about 900 nM to about 1000 nM, more preferably from about 5 nM to about 300 nM, most preferably about 100 nM, as determined by flow cytometry.

In some aspects, the anti-CD3 antibodies and fragments thereof of the invention are monovalent constructs having an in vitro binding affinity (K_d) to human T cells expressing human CD3 that is from about 5 nM to about 1000 nM preferably from about 5 nM to about 50 nM, from about 50 nM to about 100 nM, from about 100 nM to about 200 nM, from about 200 nM to about 300 nM, from about 300 nM to about 400 nM, from about 400 nM to about 500 nM, from about 500 nM to about 600 nM, from about 600 nM to about 700 nM, from about 700 nM to about 800 nM, from about 800 nM to about 900 nM, and from about 900 nM to about 1000 nM, more preferably from about 100 nM to about 250 nM, most preferably about 250 nM, as determined by flow cytometry.

In one aspect, the anti-CD3 antibodies and fragments thereof described herein compete with commercial CD3 antibody SP34-2 (BD Biosciences 551916) for binding of CD3, as determined by a competition binding assay using an AlexaFluor 488-conjugated SP34-2 antibody on primary human T cells measured using flow cytometry.

In one aspect, the anti-CD3 antibodies and fragments thereof exhibit no post-translational modification, including no oxidation, no deamidation, and no aspartate isomerization, as determined by peptide mapping.

In one aspect, the anti-CD3 antibodies and fragments thereof are effective in activating T cells and inducing CD69 expression to a similar degree as SP34-2 in human and cynomolgus monkey T cells and cOKT3 in human T cells, as determined by a T cell based assay using flow cytometry.

In one aspect, the anti-CD3 antibodies and fragments thereof described herein have a total enthalpy of unfolding of about 400 kcal/mol or more, about 410 kcal/mol or more, about 420 kcal/mol or more, about 430 kcal/mol or more, about 440 kcal/mol or more, about 45 kcal/mol or more, about 460 kcal/mol or more, about 470 kcal/mol or more, about 480 kcal/mol or more, about 490 kcal/mol or more, about 500 kcal/mol or more, about 510 kcal/mol or more, about 520 kcal/mol or more, about 530 kcal/mol or more, about 540 kcal/mol or more, or about 550 kcal/mol or more. In certain aspects, the anti-CD3 antibodies and fragments thereof of the invention have a total enthalpy of unfolding of about 418 kcal/mol, 545 kcal/mol, about 402 kcal/mol, or about 406 kcal/mol, and the anti-CD3 antibodies are CD3B376 (IgG4 PAA), CD3B450 (IgG4 PAA), CD3B389 (IgG1sigma), and CD3B467 (IgG1sigma) molecules, respectively.

Exemplary such antibodies include CD3B311, CD3B312, CD3B313, CD3B314, CD3B315, CD3B316, CD3B317, CD3B334, CD3B376 CD3B389, CD3B450 and CD3B467, and CD3B376 and CD3B450 engineered into a monovalent format.

The anti-CD3 antibodies or antigen-binding fragments of the invention can occur in a variety of forms, but will include one or more of the antibody variable domain segments or CDRs shown in Table 7A and engineered variants thereof, for example, those shown or described in Table 9 and Table 10, and the accompanying descriptions thereof.

The invention also provides an anti-CD3 antibody, or an antigen-binding fragment thereof, comprising a heavy chain comprising a HCDR1, a HCDR2, and a HCDR3 of any one of the antibodies described in Table 7B. The invention also provides an anti-CD3 antibody or an antigen-binding fragment thereof, comprising a heavy chain comprising a HCDR1, a HCDR2, and a HCDR3 of any one of the antibodies described in Table 7B and a light chain comprising a LCDR1, a LCDR2, and a LCDR3 of any one of the antibodies described in Table 7B. In some embodiments, the anti-CD3 antibody or an antigen-binding fragment thereof of the invention competes for binding to CD3 with an antibody or antigen-binding fragment thereof comprising a heavy chain comprising a HCDR1, a HCDR2, and a HCDR3 of any one of the antibodies described in Table 7B and a light chain comprising a LCDR1, a LCDR2, and a LCDR3 of any one of the antibodies described in Table 7B.

In some embodiments, the anti-CD3 antibody of an antigen-binding fragment thereof of the invention comprises the HCDR1, the HCDR2 and the HCDR3 contained within a heavy chain variable region (VH) of SEQ ID NOs:651, 652, 653, 654, 655, 687, or 656, wherein the HCDR1, the HCDR2 and the HCDR3 are defined by Chothia, Kabat, or IMGT.

In some embodiments, the anti-CD3 antibody of an antigen-binding fragment thereof of the invention comprises the LCDR1, the LCDR2 and the LCDR3 contained within a light chain variable region (VL) of SEQ ID NOs: 658, 659, 694, 660, 688, or 661, wherein the LCDR1, the LCDR2 and the LCDR3 are defined by Chothia, Kabat, or IMGT.

In some embodiments, the anti-CD3 antibody of an antigen-binding fragment thereof of the invention comprises:

the HCDR1 of SEQ ID NOs: 662, 665, or 666;

the HCDR2 of SEQ ID NOs: 663, 689, or 695; and

the HCDR3 of SEQ ID NOs: 664.

In some embodiments, the anti-CD3 antibody of an antigen-binding fragment thereof of the invention comprises:

the LCDR1 of SEQ ID NOs: 773, 710, 674 or 671;

the LCDR2 of SEQ ID NOs: 669 or 673; and

the LCDR3 of SEQ ID NOs: 670.

In some embodiments, the anti-CD3 antibody of an antigen-binding fragment thereof of the invention comprises:

the HCDR1 of SEQ ID NOs: 662, 665, or 666;

the HCDR2 of SEQ ID NOs: 663, 689, or 695;

the HCDR3 of SEQ ID NOs: 664;

the LCDR1 of SEQ ID NOs: 773, 710, 674 or 671;

the LCDR2 of SEQ ID NOs: 669 or 673; and

the LCDR3 of SEQ ID NOs: 670.

In some embodiments, the anti-CD3 antibody of an antigen-binding fragment thereof of the invention comprises the HCDR1, the HCDR2 and the HCDR3 of:

SEQ ID NOs: 662, 663, and 664, respectively;

SEQ ID NOs: 662, 695, and 664, respectively;

SEQ ID NOs: 665, 663, and 664, respectively;

SEQ ID NOs: 665, 695, and 664, respectively;

SEQ ID NOs: 662, 689, and 664, respectively;

SEQ ID NOs: 666, 663, and 664, respectively;

SEQ ID NOs: 666, 695, and 664, respectively

SEQ ID NOs: 665, 689, and 664, respectively; or

SEQ ID NOs: 666, 689, 664, respectively.

In some embodiments, the anti-CD3 antibody of an antigen-binding fragment thereof of the invention comprises the LCDR1, the LCDR2 and the LCDR3 of:

SEQ ID NOs: 773, 669, and 670, respectively;

SEQ ID NOs: 773, 673, and 670, respectively;

SEQ ID NOs: 710, 673, and 670, respectively;

SEQ ID NOs: 674, 673, and 670, respectively;

SEQ ID NOs: 671, 673, and 690, respectively;

SEQ ID NOs: 773, 673, and 690, respectively;

SEQ ID NOs: 671, 669, and 670, respectively; or

SEQ ID NOs: 776, 673, and 670, respectively.

In some embodiments, the anti-CD3 antibody of an antigen-binding fragment thereof of the invention comprises the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 662, 663, 664, 773, 669, and 670, respectively.

In some embodiments, the anti-CD3 antibody of an antigen-binding fragment thereof of the invention comprises the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 665, 663, 664, 773, 669, and 670, respectively.

In some embodiments, the anti-CD3 antibody of an antigen-binding fragment thereof of the invention comprises the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 666, 663, 664, 773, 669, and 670, respectively.

In some embodiments, the anti-CD3 antibody of an antigen-binding fragment thereof of the invention comprises a heavy chain (HC) sequence of SEQ ID NO: 709, 640, 641, 642, 643, 675, or 644 and/or a light chain (LC) sequence of SEQ ID NO: 645, 716, 649, 676, 677, or 650. In some embodiments, the anti-CD3 antibody of an antigen-binding fragment thereof of the invention comprises a HC having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:709, 640, 641, 642, 643, 675, or 644, and a LC having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:645, 716, 649, 676, 677, or 650. In some embodiments, the anti-CD3 antibody of an antigen-binding fragment thereof of the invention comprises a HC having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 709, 640, 641, 642, 643, 675, or 644, and a LC having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:645, 716, 649, 676, 677 or 650, wherein the variation in sequence does not occur in a CDR region.

In some embodiments, the anti-CD3 antibody of an antigen-binding fragment thereof of the invention comprises a heavy chain variable region (VH) sequence of SEQ ID NO:651, 652, 657, 653, 654, 655, 687, or 656 and/or a light chain variable region (VL) sequence of SEQ ID NO:658, 659, 694, 660, 688, 678, or 661. In some embodiments, the anti-CD3 antibody of an antigen-binding fragment thereof of the invention comprises a VH having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 651, 652, 657, 653, 654, 655, 687, or 656, and a VL having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOS 658, 659, 694, 660, 688, 678, or 661. In some embodiments, the anti-CD3 antibody of an antigen-binding fragment thereof of the invention comprises a VH having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 651, 652, 657, 653, 654, 655, 687, or 656, and a VL having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% or more, such as 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:658, 659, 694, 660, 688, 678, or 661, wherein the variation in sequence does not occur in a CDR region.

PSMA-Specific Antibodies

The antibodies and fragments thereof that bind to PSMA bind to the chimpanzee target antigen. In one embodiment, the antibodies and fragments thereof bind to the human and macaque PSMA target antigens with affinities within 5-fold of each other. In other words, the difference in antibody binding is less than a multiple of 5. In this case, the identical antibody molecule can be used both for preclinical evaluation of safety, activity and/or pharmacokinetic profile of PSMA in primates and as a drug in humans. Put in other words, the same PSMA-specific molecule can be used in preclinical animal studies as well as in clinical studies in humans. This leads to highly comparable results and a much-increased predictive power of the animal studies compared to species-specific surrogate molecules. Since the PSMA domain is cross-species specific, i.e. reactive with the human and macaque antigens, the antibody or fragments thereof of the invention can be used both for preclinical evaluation of safety, activity and/or pharmacokinetic profile of these binding domains in primates and—in the identical form—as drug in humans.

The present invention also provides for multispecific antibodies that specifically bind to PSMA. According to the invention, a bispecific, i.e. bifunctional, antibody can be used to engage two different therapeutic targets or perform two distinct functions. Such antibodies can be used for example to recruit an immune effector cell, e.g. T- or NK-cell, towards a particular target cell. Various antibody-fragment based molecules are known and under investigation, for example for cancer therapy.

The present invention also provides for a PSMA×“effector antigen” bispecific antibody. In one embodiment, the effector antigen of the PSMA×“effector antigen” bispecific antibody is CD3. It has been found in the present invention that it is possible to generate a PSMA×CD3 bispecific antibody wherein the identical molecule can be used in preclinical animal testing, as well as clinical studies and even in therapy in human. This is due to the identification of the PSMA×CD3 bispecific antibody, which, in addition to binding to human PSMA and human CD3, respectively, also binds to the homologs of antigens of chimpanzee and macaques. The PSMA×CD3 bispecific antibody of the invention can be used as a therapeutic agent against various diseases, including, but not limited, to cancer. In view of the above, the need to construct a surrogate target PSMA×CD3 bispecific antibody for testing in a phylogenetically distant (from humans) species disappears. As a result, the identical molecule can be used in animal preclinical testing as is intended to be administered to humans in clinical testing as well as following market approval and therapeutic drug administration.

In some embodiments described herein, the isolated antibody or antibody fragment thereof specifically binding PSMA has one, two, three, four or five of the following properties:

- a. binds Pan troglodytes PSMA extracellular domain (ECD) with an equilibrium dissociation constant (K_D) of 25 nM or less, wherein the K_Dis measured using ProteOn XPR36 system at +25° C.,
- b. binds LNCaP cells with a calculated EC50 of 20 nM or less and binds Macaca fascicularis PSMA-expressing HEK cells with a calculated EC50 of 40 nM or less, wherein the difference in calculated EC50 between binding LNCaP cells and binding Macaca fascicularis PSMA-expressing HEK cells is less than 5-fold, and wherein the calculated EC50 is measured in a whole cell binding assay at 0° C. using flow cytometry,
- c. binds recombinant PSMA ECD from human (SEQ ID NO:55), Pan troglodytes (SEQ ID NO:52) and Macaca fascicularis (SEQ ID NO:53) with an equilibrium dissociation constant (K_D) of 12 nM or less, wherein the K_Dis measured using Proteon surface plasmon resonance assay ProteOn XPR36 system at +25° C.;
- d. displays T-cell mediated killing of LNCaP cells, C42 cells, human PSMA-expressing HEK cells or Macaca fascicularis PSMA-expressing HEK cells when paired in a bispecific antibody with anti-CD3 antibody, wherein the T-cell mediated killing is measured by Chromium-51 or by caspase 3/7 activation assay, or
- e. recognizes a conformational epitope wherein the epitope is comprised of residues I138, F235, P237, G238, D244, Y299, Y300, Q303, K304, E307, and K324-P326 of human PSMA (SEQ ID NO:51)

Exemplary such antibodies or fragments thereof are PSMA antibodies PSMB119, PSMB120, PSMB121, PSMB122, PSMB123, PSMB87, PSMB126, PSMB127, PSMB128, PSMB129, PSMB130, PSMB120, PSMB121, PSMB122, PSMB123, PSMB127, PSMB128, PSMB130, PSMB344, PSMB345, PSMB346, PSMB347, PSMB349, PSMB358, PSMB359, PSMB360, PSMB361, PSMB362, PSMB363, and PSMB365 described herein.

In some embodiments of the invention described herein, the antibody specifically binding PSMA of the invention comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs:56, 57, 58, 59, 60, and 61, respectively.

In some embodiments of the invention described herein, the antibody specifically binding PSMA of the invention comprises a heavy chain variable region (VH) of SEQ ID NOs: 6, 7, 8, 110, 112, 114, 116, 118, 120, 121, 123, 125, 126, 128, 130, or 681. In some embodiments of the invention described herein, the antibody specifically binding PSMA of the invention comprises a light chain variable region (VL) of SEQ ID NOs: 9, 111, 113, 115, 117, 119, 122, 124, 127, 129, 131, or 682.

In some embodiments of the invention described herein, the antibody specifically binding PSMA of the invention comprises a heavy chain sequence of SEQ ID NOs: 12, 13, 132, 134, 136, 138, 140, 141, 143, 145, 146, 148, 150, 151, or 679.

In some embodiments of the invention described herein, the antibody specifically binding PSMA of the invention comprises a light chain sequence of SEQ ID NOs: 14, 15, 75, 133, 135, 137, 139, 142, 144, 147, 149 or 680.

CD33-Specific Antibodies

The CD33-specific antibodies of the invention possess one or more desirable functional properties, including but not limited to high-affinity binding to CD33 and/or CD3, high specificity to CD33 and/or CD3, and the ability to treat or prevent cancer when administered alone or in combination with other anti-cancer therapies.

In certain embodiments, the isolated monoclonal antibodies or antigen-binding fragments thereof bind the C2 domain of CD33. In certain embodiments, the isolated monoclonal antibodies or antigen-binding fragments thereof bind the V domain of CD33. The full length human CD33 is provided by Uniprot P20138 (SEQ ID NO:244).

As used herein, an antibody that “specifically binds to CD33” refers to an antibody that binds to a CD33, preferably a human CD33, preferably the C2 domain of CD33, with a KD of 1×10⁻⁷M or less, preferably 1×10⁻⁸M or less, more preferably 5×10⁻⁹M or less, 1×10⁻⁹M or less, 5×10⁻¹⁰M or less, or 1×10⁻¹⁰M or less.

The antibodies or antigen-binding fragments described herein can occur in a variety of forms, but will include one or more of the antibody CDRs shown in Table 39 and 40.

Described herein are recombinant antibodies and antigen-binding fragments that specifically bind to CD33. In some embodiments, the CD33-specific antibodies or antigen-binding fragments are human IgG, or derivatives thereof. While the CD33-specific antibodies or antigen-binding fragments exemplified herein are human, the antibodies or antigen-binding fragments exemplified may be chimerized.

In some embodiments are provided an CD33-specific antibody, or an antigen-binding fragment thereof, comprising a heavy chain comprising a CDR1, a CDR2, and a CDR3 of any one of the antibodies described in Table 39. In some embodiments are provided an CD33-specific antibody, or an antigen-binding fragment thereof, comprising a heavy chain comprising a CDR1, a CDR2, and a CDR3 of any one of the antibodies described in Table 39 and a light chain comprising a CDR1, a CDR2, and a CDR3 of any one of the antibodies described in Table 40.

In some embodiments are provided a CD33-specific antibody, or antigen-binding fragment thereof, comprising a heavy chain variable region shown in Table 38. In some embodiments are provided a CD33-specific antibody, or antigen-binding fragment thereof, comprising a light chain variable region shown in Table 38.

The heavy chain variable domain and light chain variable domain of antibodies discussed in this section and shown in Table 38 are suitable for inclusion in bispecific constructs. For example, in some embodiments of the CD33 bispecific antibodies, the effector arm is a CD3 arm. In some embodiments of the CD33×CD3 bispecific antibody, the CD3 arm comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2, and the LCDR3 of SEQ ID NOs:662, 663, 664, 671, 673, and 670, respectively. In some embodiments of the CD33×CD3 bispecific antibody, the CD3 arm comprises the VH and VL of SEQ ID NOs: 652 and 661, respectively. In some embodiments of the CD33×CD3 bispecific antibody, the CD3 arm comprises the HC and LC of SEQ ID NOs: 640 and 676, respectively.

In some embodiments of the CD33×CD3 bispecific antibody, the CD3 arm comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2, and the LCDR3 of SEQ ID NOs:662, 663, 664, 773, 673, and 670, respectively. In some embodiments of the CD33×CD3 bispecific antibody, the CD3 arm comprises the VH and VL of SEQ ID NOs:657 and 678, respectively. In some embodiments of the CD33×CD3 bispecific antibody, the CD3 arm comprises comprises the HC and LC of SEQ ID NOs:675 and 677, respectively.

In certain embodiments, the anti-CD33 antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence at least 95% identical to SEQ ID NO:267, 260, 275, 270, 262, 258, 257, 281, 292, 291, 261, 269, 280, 259, 263, 264, 265, 266, 272, 277, 279, 284, or 285, or a light chain variable region having a polypeptide sequence at least 95% identical to SEQ ID NO:287, 314, 309, 301, 298, 297, 290, 332, 331, 302, 310, 320, 300, 304, 305, 306, 307, 317, 319, 324, or 325; and the anti-CD3 antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence at least 95% identical to SEQ ID NO:257 or 258, or a light chain variable region having a polypeptide sequence at least 95% identical to SEQ ID NO:298 or 299.

IL1RAP-Specific Antibodies

As used herein, the terms “interleukin-1 receptor accessory protein”, “IL1RAP” and “IL1-RAP” specifically include the human IL1RAP protein (SEQ ID NO:576), for example as described in GenBank Accession No. AAB84059, NCBI Reference Sequence: NP_002173.1 and UniProtKB/Swiss-Prot Accession No. Q9NPH3-1 (see also Huang et al., 1997, Proc. Natl. Acad. Sci. USA. 94 (24), 12829-12832). IL1RAP is also known in the scientific literature as IL1 R3, C3orf13, FLJ37788, IL-1 RAcP and EG3556.

The antibodies or antigen-binding fragments described herein can occur in a variety of forms, but will include one or more of the antibody CDRs shown in Table 29.

Described herein are recombinant antibodies and antigen-binding fragments that specifically bind to IL1RAP. In some embodiments, the IL1RAP-specific antibodies or antigen-binding fragments are human IgG, or derivatives thereof. While the IL1RAP-specific antibodies or antigen-binding fragments exemplified herein are human, the antibodies or antigen-binding fragments exemplified may be chimerized.

In some embodiments are provided an IL1RAP-specific antibody, or an antigen-binding fragment thereof, comprising a heavy chain comprising a CDR1, a CDR2, and a CDR3 of any one of the antibodies described in Table 29. In some embodiments are provided an IL1RAP-specific antibody, or an antigen-binding fragment thereof, comprising a heavy chain comprising a CDR1, a CDR2, and a CDR3 of any one of the antibodies described in Table 29 and a light chain comprising a CDR1, a CDR2, and a CDR3 of any one of the antibodies described in Table 29.

In some embodiments are provided a IL1RAP-specific antibody, or antigen-binding fragment thereof, comprising a heavy chain variable region of any one of the antibodies shown in Table 30. In some embodiments are provided a IL1RAP-specific antibody, or antigen-binding fragment thereof, comprising a light chain variable region of any one of the antibodies shown in Table 30. In some embodiments are provided a IL1RAP-specific antibody, or antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region of any one of the antibodies shown in Table 30.

The heavy chain variable domain and light chain variable domain of antibodies discussed in this section and shown in Table 30 are suitable for inclusion in bispecific constructs in which the targeting arm is an anti-IL1RAP arm. For example, in some embodiments of the IL1RAP bispecific antibodies, the effector arm is a CD3 arm. In some embodiments of the IL1RAP×CD3 bispecific antibody, the CD3 arm comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2, and the LCDR3 of SEQ ID NOs:662, 663, 664, 671, 673, and 670, respectively. In some embodiments of the IL1RAP×CD3 bispecific antibody, the CD3 arm comprises the V_Hand VL of SEQ ID NOs: 652 and 661, respectively. In some embodiments of the IL1RAP×CD3 bispecific antibody, the CD3 arm comprises the HC and LC of SEQ ID NOs: 640 and 676, respectively.

In some embodiments of the IL1RAP×CD3 bispecific antibody, the CD3 arm comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2, and the LCDR3 of SEQ ID NOs:662, 663, 664, 773, 673, and 670, respectively. In some embodiments of the IL1RAP×CD3 bispecific antibody, the CD3 arm comprises the VH and VL of SEQ ID NOs:657 and 678, respectively. In some embodiments of the IL1RAP×CD3 bispecific antibody, the CD3 arm comprises comprises the HC and LC of SEQ ID NOs:675 and 677, respectively.

TMEFF2-Specific Antibodies

The invention provides an isolated anti-TMEFF2 antibody or an antigen binding fragment thereof that binds to a membrane proximal region of SEQ ID NO: 629 of TMEFF2. The anti-TMEFF2 antibodies of the invention binding the membrane proximal region of TMEFF2 are not internalized by cells. While wishing not to be bound by any particular theory, it can be expected that non-internalizing anti-TMEFF2 antibodies have improved oncogenic effect mediated by antibody effector functions resulting from lack of internalization and degradation of TMEFF2 when compared to internalizing anti-TMEFF2 antibodies.

“Binds to a membrane proximal region” means that 90% of antibody epitope residues identified using hydrogen/deuterium exchange (H/D exchange) reside within the membrane proximal region of TMEFF2. The epitope residues are those which are protected by the test antibody by at least 5% difference in deuteration levels through H/D exchange. Exemplary such antibodies are TMEB675, TMEB570, TMEB674, TMEB565, TMEB762 and TMEB757 as described herein.

In some embodiments, the isolated anti-TMEFF2 antibody or the antigen binding fragment thereof binds within residues HGKCEHSINMQEPSC (SEQ ID NO: 592) or DAGYTGQHCEKKDYSVL (SEQ ID NO: 600) to the membrane proximal region of TMEFF2. An exemplary anti-TMEFF2 antibody binding within residues HGKCEHSINMQEPSC (SEQ ID NO: 592) is TMEB570. An exemplary anti-TMEFF2 antibody binding within residues DAGYTGQHCEKKDYSVL (SEQ ID NO: 600) is TMEB675. TNEB675 variants TMEB762 and TMEB757 are also expected to bind the membrane proximal region of TMEFF2 within residues DAGYTGQHCEKKDYSVL (SEQ ID NO: 600).

In an H/D exchange assay, recombinantly expressed TMEFF2 ECD is incubated in the presence or absence of the antibody in deuterated water for predetermined times resulting in deuterium incorporation at exchangeable hydrogen atoms which are unprotected by the antibody, followed by protease digestion of the protein and analyses of the peptide fragments using LC-MS. H/D exchange assay can be performed using known protocols. An exemplary protocol is described in Example 5.

The invention also provides an isolated anti-TMEFF2 antibody or an antigen binding fragment thereof, wherein the antibody or the antigen binding fragment thereof competes for binding to the membrane proximal region of TMEFF2 with a reference antibody comprising a heavy chain variable region (VH) of SEQ ID NO: 25 and a light chain variable region (VL) of SEQ ID NO: 28, the VH of SEQ ID NO: 589 and the VL of SEQ ID NO: 29, the VH of SEQ ID NO: 27 and the VL of SEQ ID NO: 30, the VH of SEQ ID NO: 589 and the VL of SEQ ID NO: 31, the VH of SEQ ID NO: 604 and the VL of SEQ ID NO: 607, or the VH of SEQ ID NO: 612 and the VL of SEQ ID NO: 613.

Competition for binding of a test antibody to the membrane proximal region of TMEFF2 with the reference antibody may be assayed in vitro using well known methods. For example, binding of MSD Sulfo-Tag™ NETS-ester-labeled test antibody to the membrane proximal region of TMEFF2 in the presence of an unlabeled reference antibody may be assessed by ELISA, or Bioacore analyses or flow cytometry may be used to demonstrate competition. The test antibody competes for binding to TMEFF2 with the reference antibody when the test antibody inhibits binding of the reference antibody to the membrane proximal region of TMEFF2 by 85% or more, for example 90% or more, or 95% or more.

The invention also provides an isolated anti-TMEFF2 antibody or an antigen binding fragment thereof comprising a heavy chain complementarity determining region 1 (HCDR1), a HCDR2, a HCDR3, a light chain complementarity determining region 1 (LCDR1), a LCDR2 and a LCDR3 of

SEQ ID NOs: 582, 584, 587, 18, 588 and 22, respectively;

SEQ ID NOs: 583, 585, 16, 19, 21, and 23, respectively;

SEQ ID NOs: 582, 586, 17, 18, 588 and 24, respectively;

SEQ ID NOs: 583, 585, 16, 18, 588 and 22, respectively; or

SEQ ID NOs: 582, 584, 587, 18, 588 and 603, respectively.

In some embodiments, the isolated anti-TMEFF2 antibody or the antigen binding fragment thereof of the invention binds to the membrane proximal region of TMEFF2 with an equilibrium dissociation constant (K_D) of about 0.4×10⁻⁹M or less, wherein the K_Dis measured using surface plasmon resonance in acetate buffer at pH 4.5-5.0 at room temperature.

In some embodiments, the isolated anti-TMEFF2 antibody or the antigen binding fragment thereof binds to the membrane proximal region of TMEFF2 with the K_Dof between about 0.1×10⁻¹⁰M and about 0.4×10⁻⁹M.

The affinity of an antibody to the membrane proximal region of TMEFF2 may be determined experimentally using any suitable method. An exemplary method utilizes ProteOn XPR36, Biacore 3000 or KinExA instrumentation, ELISA or competitive binding assays known to those skilled in the art. The measured affinity of an antibody to TMEFF2 may vary if measured under different conditions (e.g., osmolarity, pH). Thus, measurements of affinity and other binding parameters (e.g., K_D, K_on, and Koff) are typically made with standardized conditions and a standardized buffer, such as the buffer described herein. Skilled in the art will appreciate that the internal error for affinity measurements for example using Biacore 3000 or ProteOn (measured as standard deviation, SD) can typically be within 5-33% for measurements within the typical limits of detection. Therefore, the term “about” when referring to a K_Dvalue reflects the typical standard deviation in the assay. For example, the typical SD for a K_Dof 1×10⁻⁹M is up to +0.33×10⁻⁹M.

The invention also provides an isolated anti-TMEFF2 antibody or an antigen binding fragment thereof that binds to the membrane proximal region of TMEFF2, comprising a heavy chain variable region (VH) framework derived from VH3_3-23 (SEQ ID NO: 53) or VH1_1-69 (SEQ ID NO: 54).

The invention also provides an isolated anti-TMEFF2 antibody or an antigen binding fragment thereof that binds to the membrane proximal region of TMEFF2, comprising a light chain variable region (VL) framework derived from VKI_L11 (SEQ ID NO: 55) or VKIIII_A27 (SEQ ID NO: 591).

The invention also provides an isolated anti-TMEFF2 antibody or an antigen binding fragment thereof that binds to the membrane proximal region of TMEFF2, comprising a VH framework and a VL framework derived from VH3_3-23 of SEQ ID NO: 53 and VKI_L11 of SEQ ID NO: 55, respectively.

The invention also provides an isolated anti-TMEFF2 antibody or an antigen binding fragment thereof that binds to the membrane proximal region of TMEFF2, comprising a VH framework and a VL framework derived from VH1_1-69 of SEQ ID NO: 54 and VKIII_A27 of SEQ ID NO: 591, respectively.

The invention also provides an isolated anti-TMEFF2 antibody or an antigen binding fragment thereof that binds to the membrane proximal region of TMEFF2, comprising a VH framework and a VL framework derived from VH1_1-69 of SEQ ID NO: 54 and VKI_L11 of SEQ ID NO: 55, respectively.

Antibodies comprising heavy or light chain variable regions “derived from” a particular framework or germline sequence refer to antibodies obtained from a system that uses human germline immunoglobulin genes, such as from transgenic mice, rats or chicken or from phage display libraries as discussed herein. An antibody containing particular framework derived from germline sequence may contain amino acid differences as compared to the sequence it was derived from, due to, for example, naturally-occurring somatic mutations or intentional substitutions.

The invention also provides an isolated anti-TMEFF2 antibody or an antigen binding fragment thereof, comprising a HCDR1, a HCDR2, a HCDR3, a LCDR1, a LCDR2 and a LCDR3 of SEQ ID NOs: 582, 584, 587, 18, 588 and 22, respectively.

In some embodiments, the isolated anti-TMEFF2 antibody or the antigen-binding fragment thereof comprises a VH of SEQ ID NO: 25 and a VL of SEQ ID NO: 28.

In some embodiments, the VH is encoded by a polynucleotide of SEQ ID NO: 39 and the VL is encoded by a polynucleotide of SEQ ID NO: 42.

In some embodiments, the isolated anti-TMEFF2 antibody or the antigen binding fragment thereof comprises a HC of SEQ ID NO: 32 and a LC of SEQ ID NO: 35.

In some embodiments, the HC is encoded by a polynucleotide of SEQ ID NO: 46 and the VL is encoded by a polynucleotide of SEQ ID NO: 49.

The invention also provides an isolated anti-TMEFF2 antibody or an antigen binding fragment thereof comprising a HCDR1, a HCDR2, a HCDR3, a LCDR1, a LCDR2 and a LCDR3 of SEQ ID NOs: 583, 585, 16, 19, 21 and 23, respectively.

In some embodiments, the isolated anti-TMEFF2 antibody or the antigen-binding fragment thereof comprises a VH of SEQ ID NO: 589 and a VL of SEQ ID NO: 29.

In some embodiments, the VH is encoded by a polynucleotide of SEQ ID NO: 40 and the VL is encoded by a polynucleotide of SEQ ID NO: 43.

In some embodiments, the isolated anti-TMEFF2 antibody or the antigen binding fragment thereof comprises a HC of SEQ ID NO: 33 and a LC of SEQ ID NO: 36.

In some embodiments, the HC is encoded by a polynucleotide of SEQ ID NO: 47 and the LC is encoded by a polynucleotide of SEQ ID NO: 50.

In some embodiments, the isolated anti-TMEFF2 antibody or the antigen binding fragment thereof comprises a VH of SEQ ID NO: 27 and a VL of SEQ ID NO: 30.

In some embodiments, the VH is encoded by a polynucleotide of SEQ ID NO: 41 and the VL is encoded by a polynucleotide of SEQ ID NO: 44.

In some embodiments, the isolated anti-TMEFF2 antibody or the antigen binding fragment thereof comprises a HC of SEQ ID NO: 34 and a LC of SEQ ID NO: 37.

In some embodiments, the HC is encoded by a polynucleotide of SEQ ID NO: 48 and the LC is encoded by a polynucleotide of SEQ ID NO: 51.

In some embodiments, the isolated anti-TMEFF2 antibody or the antigen binding fragment thereof comprises a VH of SEQ ID NO: 589 and a VL of SEQ ID NO: 31.

In some embodiments, the VH is encoded by a polynucleotide of SEQ ID NO: 40 and the VL is encoded by a polynucleotide of SEQ ID NO: 45.

In some embodiments, the isolated anti-TMEFF2 antibody or the antigen binding fragment thereof comprises a HC of SEQ ID NO: 33 and a LC of SEQ ID NO: 38.

In some embodiments, the HC is encoded by a polynucleotide of SEQ ID NO: 47 and the LC is encoded by a polynucleotide of SEQ ID NO: 590.

In some embodiments, the isolated anti-TMEFF2 antibody or the antigen-binding fragment thereof comprises a VH of SEQ ID NO: 604 and a VL of SEQ ID NO: 607.

In some embodiments, the VH is encoded by a polynucleotide of SEQ ID NO: 618 and the VL is encoded by a polynucleotide of SEQ ID NO: 619.

In some embodiments, the isolated anti-TMEFF2 antibody or the antigen binding fragment thereof comprises a HC of SEQ ID NO: 614 and a LC of SEQ ID NO: 615.

In some embodiments, the HC is encoded by a polynucleotide of SEQ ID NO: 620 and the LC is encoded by a polynucleotide of SEQ ID NO: 621.

In some embodiments, the isolated anti-TMEFF2 antibody or the antigen binding fragment thereof comprises a VH of SEQ ID NO: 612 and a VL of SEQ ID NO: 613.

In some embodiments, the VH is encoded by a polynucleotide of SEQ ID NO: 622 and the VL is encoded by a polynucleotide of SEQ ID NO: 623.

In some embodiments, the isolated anti-TMEFF2 antibody or the antigen binding fragment thereof comprises a HC of SEQ ID NO: 616 and a LC of SEQ ID NO: 617.

In some embodiments, the HC is encoded by a polynucleotide of SEQ ID NO: 624 and the LC is encoded by a polynucleotide of SEQ ID NO: 625.

In some embodiments, the isolated anti-TMEFF2 antibody or the antigen binding fragment thereof is a multispecific antibody.

In some embodiments, the isolated anti-TMEFF2 antibody or the antigen binding fragment thereof is a bispecific antibody.

In some embodiments, the isolated anti-TMEFF2 bispecific antibody or the antigen binding fragment thereof binds a T cell antigen.

In some embodiments, the isolated anti-TMEFF2 bispecific antibody or the antigen binding fragment thereof binds CD3.

In some embodiments, the isolated anti-TMEFF2 bispecific antibody or the antigen-binding fragment thereof binds CD3 epsilon.

The VH, the VL, the HCDR, the LCDR, the HC and the LC sequences of exemplary anti-TMEFF2 antibodies of the invention are shown in Tables 60-67.

Although the embodiments illustrated in the Examples comprise pairs of variable domains, one from a heavy chain and one from a light chain, a skilled artisan will recognize that alternative embodiments may comprise single heavy or light chain variable domains. The single variable domain may be used to screen for variable domains capable of forming a two-domain specific antigen-binding fragment capable of binding to TMEFF2. The screening may be accomplished by phage display screening methods using for example hierarchical dual combinatorial approach disclosed in Int. Patent Publ. No. WO1992/01047. In this approach, an individual colony containing either a VH or a VL chain clone is used to infect a complete library of clones encoding the other chain (VL or VH), and the resulting two-chain specific antigen-binding domain is selected in accordance with phage display techniques using known methods and those described herein. Therefore, the individual VH and VL polypeptide chains are useful in identifying additional anti-TMEFF2 antibodies using the methods disclosed in Int. Patent Publ. No. WO1992/01047.

Bispecific Anti-TMEFF2/Anti-CD3 Antibodies

The invention also provides an isolated bispecific anti-TMEFF2/anti-CD3 antibody or an antigen binding fragment thereof comprising a first domain that binds TMEFF2 and a second domain that binds CD3, wherein the antibody competes for binding to the membrane proximal region of TMEFF2 with a reference antibody comprising a heavy chain variable region (VH) of SEQ ID NO: 25 and a light chain variable region (VL) of SEQ ID NO: 28, the VH of SEQ ID NO: 589 and the VL of SEQ ID NO: 29, the VH of SEQ ID NO: 27 and the VL of SEQ ID NO: 30, the VH of SEQ ID NO: 589 and the VL of SEQ ID NO: 31, the VH of SEQ ID NO: 604 and the VL of SEQ ID NO: 607, or the VH of SEQ ID NO: 612 and the VL of SEQ ID NO: 613.

In some embodiments, the isolated bispecific anti-TMEFF2/anti-CD3 antibody or an antigen binding fragment thereof binds the membrane proximal region of TMEFF2 with a dissociation constant (K_D) of about 0.4×10⁻⁹M or less, wherein the K_Dis measured using surface plasmon resonance in acetate buffer at pH 4.5-5.0 at room temperature.

In some embodiments, the isolated bispecific anti-TMEFF2/anti-CD3 antibody or the antigen binding fragment thereof binds the membrane proximal region TMEFF2 with the K_Dof between about 0.1×10⁻¹⁰M and about 0.4×10⁻⁹M.

The invention also provides an isolated bispecific anti-TMEFF2/anti-CD3 antibody or an antigen binding fragment thereof, wherein the first domain comprises a HCDR1, a HCDR2, a HCDR3, a LCDR1, a LCDR2 and a LCDR3 of

SEQ ID NOs: 582, 584, 587, 18, 588 and 22, respectively;

SEQ ID NOs: 583, 585, 16, 19, 21 and 23, respectively;

SEQ ID NOs: 582, 586, 17, 18, 588 and 24, respectively; or

SEQ ID NOs: 583, 585, 16, 18, 588 and 22, respectively; or

SEQ ID NOs: 582, 584, 587, 18, 588 and 603, respectively.

The invention also provides an isolated bispecific anti-TMEFF2/anti-CD3 antibody or an antigen binding fragment thereof, wherein the second domain comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 662, 663, 664, 671, 673, and 690, respectively.

The invention also provides an isolated bispecific anti-TMEFF2/anti-CD3 antibody or an antigen binding fragment thereof, wherein the first domain comprises:

the VH of SEQ ID NO: 25 and the VL of SEQ ID NO: 28;

the VH of SEQ ID NO: 589 and the VL of SEQ ID NO: 29;

the VH of SEQ ID NO: 27 and the VL of SEQ ID NO: 30;

the VH of SEQ ID NO: 589 and the VL of SEQ ID NO: 31;

the VH of SEQ ID NO: 604 and the VL of SEQ ID NO: 607; or

the VH of SEQ ID NO: 612 and the VL of SEQ ID NO: 613.

The invention also provides an isolated bispecific anti-TMEFF2/anti-CD3 antibody or an antigen binding fragment thereof, wherein the second domain comprises:

the VH of SEQ ID NO: 652 and the VL of SEQ ID NO: 661.

In some embodiments, the second domain comprises the VH of SEQ ID NO:657 and the VL of SEQ ID NO: 658.

the first domain comprises a HCDR1, a HCDR2, a HCDR3, a LCDR1, a LCDR2 and a LCDR3 of SEQ ID NOs: 582, 584, 587, 18, 588 and 22, respectively, and the second domain comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 662, 663, 664, 671, 673, and 690, respectively;

the first domain comprises a VH of SEQ ID NO: 25 and a VL of SEQ ID NO: 28, and the second domain comprises the VH of SEQ ID NO: 652 and the VL of SEQ ID NO: 661;

the first domain comprises a VH of SEQ ID NO: 25 and a VL of SEQ ID NO: 28, and the second domain comprises the VH of SEQ ID NO: 657 and the VL of SEQ ID NO: 678;

the bispecific anti-TMEFF2/anti-CD3 antibody or the antigen binding fragment thereof comprises a HC1 of SEQ ID NO: 32, a LC1 of SEQ ID NO: 35, a HC2 of SEQ ID NO: 640 and a LC2 of SEQ ID NO: 676; and/or

the bispecific anti-TMEFF2/anti-CD3 antibody or the antigen binding fragment thereof comprises a HC1 of SEQ ID NO: 32, a LC1 of SEQ ID NO: 35, a HC2 of SEQ ID NO: 675 and a LC2 of SEQ ID NO: 677.

the first domain comprises a HCDR1, a HCDR2, a HCDR3, a LCDR1, a LCDR2 and a LCDR3 of SEQ ID NOs: 583, 585, 16, 19, 21, and 23, respectively, and the second domain comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 662, 663, 664, 671, 673, and 690, respectively;

the first domain comprises a VH of SEQ ID NO: 589 and a VL of SEQ ID NO: 29, and the second domain comprises the VH of SEQ ID NO: 652 and the VL of SEQ ID NO: 661;

the first domain comprises a VH of SEQ ID NO: 589 and a VL of SEQ ID NO: 29, and the second domain comprises the VH of SEQ ID NO: 657 and the VL of SEQ ID NO: 678 and/or

the bispecific anti-TMEFF2/anti-CD3 antibody or the antigen binding fragment thereof comprises a HC1 of SEQ ID NO: 33, a LC1 of SEQ ID NO: 36, a HC2 of SEQ ID NO: 640 and a LC2 of SEQ ID NO: 676;

the bispecific anti-TMEFF2/anti-CD3 antibody or the antigen binding fragment thereof comprises a HC1 of SEQ ID NO: 33, a LC1 of SEQ ID NO: 36, a HC2 of SEQ ID NO: 675 and a LC2 of SEQ ID NO: 677.

the first domain comprises a HCDR1, a HCDR2, a HCDR3, a LCDR1, a LCDR2 and a LCDR3 of SEQ ID NOs: 582, 586, 17, 18, 588 and 24, respectively, and the second domain comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 662, 663, 664, 671, 673, and 690, respectively;

the first domain comprises a VH of SEQ ID NO: 27 and a VL of SEQ ID NO: 30, and the second domain comprises the VH of SEQ ID NO: 652 and the VL of SEQ ID NO: 661;

the first domain comprises a VH of SEQ ID NO: 27 and a VL of SEQ ID NO: 30, and the second domain comprises the VH of SEQ ID NO: 657 and the VL of SEQ ID NO: 678, and/or

the bispecific anti-TMEFF2/anti-CD3 antibody or the antigen binding fragment thereof comprises a HC1 of SEQ ID NO: 34, a LC1 of SEQ ID NO: 37, a HC2 of SEQ ID NO: 640 and a LC2 of SEQ ID NO: 676;

the bispecific anti-TMEFF2/anti-CD3 antibody or the antigen binding fragment thereof comprises a HC1 of SEQ ID NO: 34, a LC1 of SEQ ID NO: 37, a HC2 of SEQ ID NO: 675 and a LC2 of SEQ ID NO: 677.

the first domain comprises a HCDR1, a HCDR2, a HCDR3, a LCDR1, a LCDR2 and a LCDR3 of SEQ ID NOs: 583, 585, 16, 18, 588 and 22, respectively, and the second domain comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 662, 663, 664, 671, 673, and 690, respectively;

the first domain comprises a VH of SEQ ID NO: 589 and a VL of SEQ ID NO: 31, and the second domain comprises the VH of SEQ ID NO: 652 and the VL of SEQ ID NO: 661;

the first domain comprises a VH of SEQ ID NO: 589 and a VL of SEQ ID NO: 31, and the second domain comprises the VH of SEQ ID NO: 657 and the VL of SEQ ID NO: 678; and/or

the bispecific anti-TMEFF2/anti-CD3 antibody or the antigen binding fragment thereof comprises a HC1 of SEQ ID NO: 33, a LC1 of SEQ ID NO: 38, a HC2 of SEQ ID NO:640 and a LC2 of SEQ ID NO: 676;

the bispecific anti-TMEFF2/anti-CD3 antibody or the antigen binding fragment thereof comprises a HC1 of SEQ ID NO: 33, a LC1 of SEQ ID NO: 38, a HC2 of SEQ ID NO:675 and a LC2 of SEQ ID NO: 677.

the first domain comprises a HCDR1, a HCDR2, a HCDR3, a LCDR1, a LCDR2 and a LCDR3 of SEQ ID NOs: 582, 584, 587, 18, 588 and 603, respectively, and the second domain comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 662, 663, 664, 671, 673, and 690, respectively;

the first domain comprises a VH of SEQ ID NO: 604 and a VL of SEQ ID NO: 607, and the second domain comprises the VH of SEQ ID NO: 652 and the VL of SEQ ID NO: 661;

the first domain comprises a VH of SEQ ID NO: 604 and a VL of SEQ ID NO: 607, and the second domain comprises the VH of SEQ ID NO: 657 and the VL of SEQ ID NO: 678; and/or

the bispecific anti-TMEFF2/anti-CD3 antibody or the antigen binding fragment thereof comprises a HC1 of SEQ ID NO: 614, a LC1 of SEQ ID NO: 615, a HC2 of SEQ ID NO: 640 and a LC2 of SEQ ID NO: 676;

the bispecific anti-TMEFF2/anti-CD3 antibody or the antigen binding fragment thereof comprises a HC1 of SEQ ID NO: 614, a LC1 of SEQ ID NO: 615, a HC2 of SEQ ID NO: 675 and a LC2 of SEQ ID NO: 677.

the first domain comprises a VH of SEQ ID NO: 612 and a VL of SEQ ID NO: 613, and the second domain comprises the VH of SEQ ID NO: 652 and the VL of SEQ ID NO: 661;

the first domain comprises a VH of SEQ ID NO: 612 and a VL of SEQ ID NO: 613, and the second domain comprises the VH of SEQ ID NO: 657 and the VL of SEQ ID NO: 678; and/or

the bispecific anti-TMEFF2/anti-CD3 antibody or an antigen binding fragment thereof comprises a HC1 of SEQ ID NO: 616, a LC1 of SEQ ID NO: 617, a HC2 of SEQ ID NO: 640 and a LC2 of SEQ ID NO: 676;

the bispecific anti-TMEFF2/anti-CD3 antibody or an antigen binding fragment thereof comprises a HC1 of SEQ ID NO: 616, a LC1 of SEQ ID NO: 617, a HC2 of SEQ ID NO: 675 and a LC2 of SEQ ID NO: 677.

Embodiments

This invention provides the following non-limiting embodiments.

- 1. An isolated recombinant anti-CD3 antibody, or antigen-binding fragment thereof, comprising:
  - a) a heavy chain comprising a heavy chain complementarity determining region (HCDR) 1 comprising SEQ ID NO: 662; a HCDR2 comprising SEQ ID NO: 663; and a HCDR3 comprising SEQ ID NO: 664 and a light chain comprising a light chain complementarity determining region (LCDR) 1 comprising SEQ ID NO: 671, a LCDR2 comprising SEQ ID NO: 673, and a LCDR3 comprising SEQ ID NO: 690;
  - b) a heavy chain variable region comprising SEQ ID NO: 652 and a light chain variable region comprising SEQ ID NO: 661;
  - c) a heavy chain comprising SEQ ID NO: 640 and a light chain comprising SEQ ID NO: 676;
  - d) a heavy chain comprising a HCDR1 comprising SEQ ID NO: 662; a HCDR2 comprising SEQ ID NO: 663; and a HCDR3 comprising SEQ ID NO: 664 and a light chain comprising a LCDR1 comprising SEQ ID NO: 773, a LCDR2 comprising SEQ ID NO: 673, and a LCDR3 comprising SEQ ID NO: 690;
  - e) a heavy chain variable region comprising SEQ ID NO: 657 and a light chain variable region comprising SEQ ID NO: 678; or
  - f) a heavy chain comprising SEQ ID NO: 675 and a light chain comprising SEQ ID NO: 678.
- 2. An isolated recombinant anti-CD3 antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment specifically binds Macaca fascicularis or human CD3d, or CD3e, or CD3e and CD3d with a binding affinity of about 300 nM or less.
- 3. The isolated recombinant anti-CD3 antibody or antigen-binding fragment thereof of embodiment 2, wherein the binding affinity is about 100 nM or less.
- 4. The isolated recombinant anti-CD3 antibody or antigen-binding fragment thereof of embodiment 2 or 3, wherein the binding affinity is measured by flow cytometry or by Proteon surface plasmon resonance assay ProteOn XPR36 system at +25° C.
- 5. The isolated recombinant anti-CD3 antibody or antigen-binding fragment thereof of any one of the previous embodiments, wherein the antibody or antigen-binding fragment has one, two, three, or four of the following properties:
  - a) binds human and Macaca fascicularis CD3+T lymphocytes with a calculated EC50 of 300 nM or less and binds Macaca fascicularis CD3-expressing HEK cells with a calculated EC50 of 300 nM or less, wherein the difference in calculated EC50 between binding CD3+T lymphocytes and binding Macaca fascicularis CD3-expressing HEK cells is less than 5-fold, and wherein the calculated EC50 is measured in a whole cell binding assay at 0° C. using flow cytometry;
  - b) binds recombinant CD3d from human (SEQ ID NO:691), or binds recombinant CD3e from human (SEQ ID NO:636), or binds recombinant CD3d from Macaca fascicularis (SEQ ID NO:692), or binds recombinant CD3e from Macaca fascicularis (SEQ ID NO:693) with an equilibrium dissociation constant (K_D) of 300 nM or less, wherein the K_Dis measured using Proteon surface plasmon resonance assay ProteOn XPR36 system at +25° C.;
  - c) binds residues 1-6 of CD3e as determined by X-ray crystallography; or
  - d) activates T cells or induces CD69 expression to a similar degree as cOKT3 or SP34-2 as determined by fluorescence-activated cell sorting assay.
- 6. The antibody or antigen-binding fragment thereof of any one of the previous embodiments comprising at least one substitution in an antibody constant domain, the at least one substitution comprising:
  - a) heavy chain substitutions K409R, F405L, or F405L and R409K;
  - b) heavy chain substitutions S228P, F234A, and L235A;
  - c) heavy chain substitutions L234A, G237A, P238S, H268A, A330S and P331S, wherein the antibody is an IgG1 isotype; or
  - d) heavy chain substitution S228P, wherein the antibody is an IgG4 isotype;
  - wherein residue numbering is according to the EU Index.
- 7. The antibody or antigen-binding fragment thereof of any one of the previous embodiments, comprising the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2, and the LCDR3 of SEQ ID NOs:662, 663, 664, 671, 673, and 690, respectively.
- 8. The antibody or antigen-binding fragment thereof of any one of the previous embodiments, comprising a heavy chain variable region (VH) and a light chain variable region (VL) of SEQ ID NOs:652 and 661, respectively.
- 9. The antibody or antigen-binding fragment thereof of any one of the previous embodiments, comprising a heavy chain sequence (HC) and a light chain sequence (LC) of SEQ ID NOs:640 and 676, respectively.
- 10. The antibody or antigen-binding fragment thereof of any one of embodiments 1-5, comprising the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2, and the LCDR3 of SEQ ID NOs:662, 663, 664, 773, 673, and 690, respectively.
- 11. The antibody or antigen-binding fragment thereof of any one of embodiments 1-5, comprising a VH and a VL of SEQ ID NOs:657 and 678, respectively.
- 12. The antibody or antigen-binding fragment thereof of any one of embodiments 1-5, comprising a HC and a LC of SEQ ID NOs:675 and 677, respectively.
- 13. An antibody or antigen-binding fragment thereof, comprising the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2, and the LCDR3 of SEQ ID NOs: 662, 663, 664, 671, 673, and 690, respectively.
- 14. An antibody or antigen-binding fragment thereof, comprising a VH and a VL of SEQ ID NOs:652 and 661, respectively.
- 15. An antibody or antigen-binding fragment thereof, comprising a HC and a LC of SEQ ID NOs:640 and 676, respectively.
- 16. An antibody or antigen-binding fragment thereof, comprising the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2, and the LCDR3 of SEQ ID NOs:662, 663, 664, 773, 673, and 690, respectively.
- 17. An antibody or antigen-binding fragment thereof, comprising a VH and a VL of SEQ ID NOs:657 and 678, respectively.
- 18. An antibody or antigen-binding fragment thereof, comprising a HC and a LC of SEQ ID NOs:675 and 677, respectively.
- 19. The antibody or antigen-binding fragment thereof of any one of the previous embodiments, wherein the antibody is human or humanized.
- 20. The antibody of embodiment 19, wherein the antibody is an IgG4 or IgG1 isotype.
- 21. The antibody of embodiment 20, comprising one, two, three, four, five, six, seven, eight, nine or ten substitutions in the antibody Fc.
- 22. The antibody of embodiment 18, comprising:
  - a) D43G, L49M, L50I, S62N, Q85E light chain substitutions;
  - b) D43G, V48L, L49M, L50I, S62N, Q85E, H89Y light chain substitutions;
  - c) R10G, R13K, V73I, R70K, T83S, L96V heavy chain substitutions;
  - d) any one of light chain substitutions D43G, V48L, L49M, L50I, S62N, Q85E, or H89Y; or
  - e) any one of heavy chain substitutions R10G, R13K, V73I, R79K, T83S, or L96V, wherein residue numbering for light chain substitutions is according to SEQ ID No: 661, and for heavy chain substitutions is according to SEQ ID No: 652.
- 23. The antibody of any one of the previous embodiments, wherein the antibody is bispecific or multispecific.
- 24. A bispecific antibody comprising a first domain that specifically binds CD3 and a second domain that specifically binds a second antigen, wherein the first domain comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2, and the LCDR3 of SEQ ID NOs:662, 663, 664, 671, 673, and 690, respectively.
- 25. The bispecific antibody of embodiment 24 wherein the first domain and second domain are an IgG4 isotype, and wherein the first or the second domain comprises S228P, F234A, L235A, F405L and R409K heavy chain substitutions and the other domain of the first or the second domain comprises S228P, F234A and L235A heavy chain substitution, wherein residue numbering is according to the EU Index.
- 26. The bispecific antibody of embodiment 24, wherein the first and/or the second domain comprises at least one substitution in a CH3 constant domain comprising a F405L, or F405L and R409K substitution, wherein residue numbering is according to the EU Index.
- 27. The bispecific antibody of embodiment 24, wherein one of the first or the second domains comprises a F405L heavy chain substitution and the other of the first or second domains comprises a K409R heavy chain substitution, wherein residue numbering is according to the EU Index.
- 28. The bispecific antibody of embodiment 24, wherein the first domain and the second domain are an IgG4 isotype, wherein one of the first or the second domains comprises a S228P heavy chain substitution and the other of the first or the second domains comprises S228P, F405L and R409K heavy chain substitutions, wherein residue numbering is according to the EU Index.
- 29. The bispecific antibody of claim 24, wherein the first domain comprises the VH and the VL of SEQ ID NOs: 652 and 661, respectively.
- 30. The bispecific antibody of claim 24, wherein the first domain comprises the HC and the LC of SEQ ID NOs: 640 and 676, respectively.
- 31. The bispecific antibody of claim 24, wherein the first domain comprises the VH and the VL of SEQ ID NOs:657 and 678, respectively.
- 32. The bispecific antibody of claim 24, wherein the first domain comprises the HC and the LC of SEQ ID NOs:675 and 677, respectively.
- 33. The bispecific antibody of claim 24, wherein the second antigen is a cell surface antigen that is expressed on a target cell other than an immune effector cell.
- 34. The bispecific antibody of claim 33, wherein the cell surface antigen is a tumor associated antigen.
- 35. The bispecific antibody of any one of claims 24-34, wherein the second antigen is CD33, IL1RAP, PSMA or TMEFF2.
- 36. The bispecific antibody of embodiment 35, wherein the first domain comprises the HCDR1, HCDR2, the HCDR3, the LCDR1, the LCDR2, and the LCDR3 of SEQ ID NOs:662, 663, 664, 671, 673, and 690, respectively; and wherein the second domain comprises the the HCDR1, HCDR2, the HCDR3, the LCDR1, the LCDR2, and the LCDR3 of SEQ ID NOs:78, 683, 80, 81, 792, and 686, respectively.
- 37. The bispecific antibody of embodiment 33, wherein the first domain comprises the VH and VL of SEQ ID NOs:652 and 661, respectively; and wherein the second domain comprises the VH and VL of SEQ ID NOs:681 and 682, respectively.
- 38. The bispecific antibody of embodiment 33, wherein the first domain comprises the HC and LC of SEQ ID NOs:640 and 676, respectively; and wherein the second domain comprises the HC and LC of SEQ ID NOs:679 and 680, respectively.
- 39. The bispecific antibody of embodiment 33, wherein the first domain comprises the HCDR1, HCDR2, the HCDR3, the LCDR1, the LCDR2, and the LCDR3 of SEQ ID NOs:662, 663, 664, 671, 673, and 690, respectively; and wherein the second domain comprises the the HCDR1, HCDR2, the HCDR3, the LCDR1, the LCDR2, and the LCDR3 of SEQ ID NOs:78, 3, 80, 81, 792 and 686, respectively.
- 40. The bispecific antibody of embodiment 33, wherein the first domain comprises the VH and VL of SEQ ID NOs:652 and 661, respectively; and wherein the second domain comprises the VH and VL of SEQ ID NOs 8 and 682, respectively.
- 41. The bispecific antibody of embodiment 33, wherein the first domain comprises the HC and LC of SEQ ID NOs:640 and 676, respectively; and wherein the second domain comprises the HC and LC of SEQ ID NOs 13 and 680, respectively.
- 42. The bispecific antibody of embodiment 33, wherein the first domain comprises the HCDR1, HCDR2, the HCDR3, the LCDR1, the LCDR2, and the LCDR3 of SEQ ID NOs:662, 663, 664, 671, 673, and 690, respectively; and wherein the second domain comprises the the HCDR1, HCDR2, the HCDR3, the LCDR1, the LCDR2, and the LCDR3 of SEQ ID NOs:78, 79, 80, 81, 82 and 83, respectively.
- 43. The bispecific antibody of embodiment 33, wherein the first domain comprises the VH and VL of SEQ ID NOs:652 and 661, respectively; and wherein the second domain comprises the VH and VL of SEQ ID NOs:116 and 117, respectively.
- 44. The bispecific antibody of embodiment 33, wherein the first domain comprises the HC and LC of SEQ ID NOs:640 and 676, respectively; and wherein the second domain comprises the HC and LC of SEQ ID NOs:138 and 139, respectively.
- 45. The bispecific antibody of embodiment 31, wherein the first domain comprises the HCDR1, HCDR2, the HCDR3, the LCDR1, the LCDR2, and the LCDR3 of SEQ ID NOs: 662, 663, 664, 671, 673, and 670, respectively; and wherein the second domain comprises the the HCDR1, HCDR2, the HCDR3, the LCDR1, the LCDR2, and the LCDR3 of SEQ ID NOs:164, 165, 166, 167, 168, and 169, respectively.
- 46. The bispecific antibody of embodiment 31, wherein the first domain comprises the VH and VL of SEQ ID NOs:652 and 661, respectively; and wherein the second domain comprises the VH and VL of SEQ ID NOs:215 and 216, respectively.
- 47. The bispecific antibody of embodiment 31, wherein the first domain comprises the HCDR1, HCDR2, the HCDR3, the LCDR1, the LCDR2, and the LCDR3 of SEQ ID NOs: 662, 663, 664, 671, 673, and 670, respectively; and wherein the second domain comprises the the HCDR1, HCDR2, the HCDR3, the LCDR1, the LCDR2, and the LCDR3 of SEQ ID NOs:349, 390, 341, 471, 513, and 555, respectively.
- 48. The bispecific antibody of embodiment 31, wherein the first domain comprises the VH and VL of SEQ ID NOs:652 and 661, respectively; and wherein the second domain comprises the VH and VL of SEQ ID NOs:267 and 306, respectively.
- 49. The bispecific antibody of embodiment 31, wherein the first domain comprises the HCDR1, HCDR2, the HCDR3, the LCDR1, the LCDR2, and the LCDR3 of SEQ ID NOs: 662, 663, 664, 671, 673, and 670, respectively; and wherein the second domain comprises the HCDR1, HCDR2, the HCDR3, the LCDR1, the LCDR2, and the LCDR3 of SEQ ID NOs:363, 404, 445, 485, 527, and 569, respectively.
- 50. The bispecific antibody of embodiment 31, wherein the first domain comprises the VH and VL of SEQ ID NOs:652 and 661, respectively; and wherein the second domain comprises the VH and VL of SEQ ID NOs:281 and 320, respectively.
- 51. A pharmaceutical composition comprising the antibody of any one of the previous embodiments and a pharmaceutically acceptable carrier.
- 52. A polynucleotide encoding the antibody of any one of the embodiments 1-50.
- 53. A vector comprising the polynucleotide of embodiment 52.
- 54. A host cell comprising the vector of embodiment 53.
- 55. A method of producing the antibody of any one of embodiments 1-50, comprising culturing the host cell of embodiment 52 in conditions that the antibody is expressed, and recovering the antibody produced by the host cell.
- 56. A method of treating a cancer in a subject, comprising administering a therapeutically effective amount of the isolated antibody of any one of embodiments 1-50 to the subject in need thereof for a time sufficient to treat the cancer.
- 57. The method of embodiment 56, wherein the cancer is a solid tumor or a hematological malignancy.
- 58. The method of embodiment 57, wherein the solid tumor is a prostate cancer, a colorectal cancer, a gastric cancer, a clear cell renal carcinoma, a bladder cancer, a lung cancer, a squamous cell carcinoma, a glioma, a breast cancer, a kidney cancer, a neovascular disorder, a clear cell renal carcinoma (CCRCC), a pancreatic cancer, a renal cancer, a urothelial cancer or an adenocarcinoma to the liver.
- 59. The method of embodiment 58, wherein the prostate cancer is a refractory prostate cancer, a prostatic intraepithelial neoplasia, an androgen independent prostate cancer, or a malignant prostate cancer.
- 60. The method of embodiment 57, wherein the hematological malignancy is acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), acute lymphocytic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), chronic myeloid leukemia (CIVIL) or blastic plasmacytoid dendritic cell neoplasm (DPDCN).
- 61. The method of any one of embodiments 56-60, wherein the antibody is administered in combination with a second therapeutic agent.
- 62. The antibody of any one of embodiments 1-50 for use in therapy.
- 63. An anti-idiotypic antibody binding to the antibody of any one of embodiments 1-50.

EXAMPLES
1 De Novo Generation and Functional Characterization of Anti-CD3 mAbs
1-1. Immunization of OmniRats® with CD3 Antigens to Generate CD3 Monoclonal Antibodies

OmniRats® were immunized with proprietary vectors (Aldevron, Fargo, North Dakota, USA) encoding for: human CD3e and human CD3d; cynomolgus CD3e and cynomolgus CD3d. Animals received alternating boosts with human and cynomolgus DNA. From the 6th application on, the animals received optimized vectors with the same insert. Cells from lymph nodes were fused with the Ag8 myeloma cell line. 40 million cells from fusion BLW were put on three 96-well plates following IgM depletion. 133 million cells from fusion BLX were put on nine 96-well plates without IgM depletion.

Hybridoma supernatants from fusions BLW (with magnetic bead depletion of lymphocytes) and BLX (without magnetic bead depletion of lymphocytes) were analyzed a) by a cell-based ELISA (CELISA) on cells transiently transfected with human and cynomolgus cDNA cloned into screening vectors: pOPT-CD3e-hum-epsilon-TCE.OMT+pOPT-CD3d-hum-delta.OMT, 1:1 (pOPT-CD3e/d-hum-mix) and pcDNA3.1-CD3e-cyn-delta+pcDNA3.1-CD3d-cyn-delta, 1:1 (pcDNA3.1-CD3e/d-cyn-mix). Human and cynomolgus cDNA sequences (and corresponding amino acid sequences) are provided in Table 4. For CELISA negative control, non-transfected mammalian cells were incubated with hybridoma supernatants and detected with the secondary Bethyl antibody. For a CELISA transfection control, mammalian cells, transfected with the constructs described above, were detected with anti-tag antibody.

Hybridoma supernatants were further analyzed by flow cytometry (FACS) on CD3 positive and CD3 negative Jurkat cells: Jurkat CD3+(E6-1) and Jurkat CD3− (J.RT3-T3.5). For FACS negative control, CD3 negative Jurkat cells (J.RT3-T3.5), were incubated with dilution buffer and detected with Southern anti-rat Ig HRP and Bethyl anti-rat IgG1, 2a, 2b, 2c-HRP secondary antibodies.

The specificity of the antibodies in the hybridoma supernatants of the hybridomas from fusion BLW and fusion BLX for human and cynomolgus CD3e/d complex presented on transiently transfected cells or, in the case of the human CD3e/d complex on Jurkat CD3+(E6-1) cells, was demonstrated by CELISA on transiently transfected cells, as well as when tested in FACS on Jurkat cell lines (Table 3). No significant signal was detected for any samples in the experimental samples serving as negative controls.

TABLE 3

Test of the specificity of individual hybridoma supernatants by cell-based ELISA

(top) and by flow-cytometry (bottom). CELISA values represent the relative fluorescence

units (rfu) of each sample. FACS values represent the geometric means (geomean)

of the relative fluorescence intensities of each sample.

Cell based ELISA (relative fluorescence units)

Hybridoma supernatants incubated on cells transfected with:

Non-transfected

pOPT-CD3e/d-
pcDNA3.1-
CHO negative

hum-mix
CD3e/d-cyn-mix
control

No
Clone
Rfu
% positive
Rfu
% positive
Rfu
% positive

BLW: fusion with lymphocytes from immunization

group MR14-379 rat 1&2 (magnetic-

bead depletion of IgM+ applied)

1
BLW-2B4
689
101
230
47
30

2
BLW-2E6
231
34
121
25
20

3
BLW-3B4
867
127
320
66
24

BLX: fusion with lymphocytes from immunization

group MR14-379 rat 1&2 (without

magnetic-bead depletion of IgM+)

4
BLX-1F8
689
101
230
47
30

5
BLX-2E9
867
127
320
66
24

6
BLX-3F4
896
131
371
76
17

7
BLX-3G8
759
111
340
70
22

8
BLX-4D9
1042
153
483
99
24

9
BLX-6A2
110
16
38
8
19

Positive
682
100
488
100
—

control

Negative
25
4
42
9
—

control

Flow Cytometry (FACS)

Hybridoma supernatants incubated on Jurkat cell line

Jurkat CD3− (J.RT3.T3.5)

Jurkat CD3+ (E6-1)
negative control

No
Clone
geomean
% positive
geomean
% positive

BLW: fusion with lymphocytes from immunization

group MR14-379 rat 1&2 (magnetic-

bead depletion of IgM+ applied)

1
BLW-2B4
148034
53
4012

2
BLW-2E6
7503
3
687

3
BLW-3B4
198849
72
2884

BLX: fusion with lymphocytes from immunization

group MR14-379 rat 1&2 (without

magnetic-bead depletion of IgM+)

4
BLX-1F8
148034
53
4012

5
BLX-2E9
198849
72
2884

6
BLX-3F4
181963
66
4613

7
BLX-3G8
214697
77
3096

8
BLX-4D9
25839
9
1471

9
BLX-6A2
3385
1
1219

Positive
277338
100
2051

control

Negative
869
0
599

control

TABLE 4

CD3 sequences used for immunization

Human CD3d
NP_000723.1 (www.uniprot.org/uniprot/P04234)

(SEQ ID NO: 691)

Human CD3e
(NP_000724.1 (www.uniprot.org/uniprot/P07766)

(SEQ ID NO: 636)

Cyno CD3d
XP_001097302 (www.uniprot.org/uniprot/Q95LI8)

(SEQ ID NO: 692)

Cyno CD3e
CD3e + TCE (www.uniprot.org/uniprot/Q95LI5)

(SEQ ID NO: 693)

1-2 Cloning Anti-CD3 Antibodies

Anti-human CD3 antibodies were generated in OmniRats (OMT, Palo Alto, California, USA). Variable region (“V region”) sequences of these clones were extracted from the genomic sequences and analyzed. All sequences obtained were either human IgG heavy chain or lambda light chain and the sequences, especially LC, showed high homology. Aligning the sequences to germline showed some mutations in the framework (FIG. 1). The V-region DNA sequences were synthesized and cloned into mammalian expression vectors, heavy chain sequences into human IgG1 vector and light chain sequences into human lambda vector. Sequences are shown in Tables 6 and 7. Seven mAbs were assigned protein identifiers (Table 5A).

CD3B312 was chosen as the most representative clone and the heavy chain sequences were cloned into human IgG1sigma and IgG4 PAA with S228P, F234A, L235A mutations and assigned protein identifiers as shown in Table 5B. These were used to generate bispecific antibodies and to demonstrate T cell redirection functionality through cytotoxicity.

TABLE 5A

Peptide ID and protein ID of clones

clone ID
HC peptide ID
LC peptide ID
protein ID

BLW-2B4
CD3H218
CD3L123
CD3B311

BLW-2E6
CD3H219
CD3L124
CD3B312

BLW-3B4
CD3H218
CD3L125
CD3B313

BLX-1F8
CD3H220
CD3L126
CD3B314

BLX-2E9
CD3H221
CD3L124
CD3B315

BLX-3F4
CD3H222
CD3L124
CD3B316

BLX-3G8
CD3H223
CD3L124
CD3B317

TABLE 5B

CD3B312 was chosen as the most representative clone and the

heavy chain sequences were cloned into human IgG1 sigma and

IgG4 PAA with mutations and assigned protein identifiers.

IgG1
IgG1sigma
IgG4PAA

CD3B312
CD3B337
CD3B373

TABLE 6

Heavy chain and light chain sequences of 7 monoclonal CD3 antibodies

Protein

SEQ ID

SEQ ID

AA ID
Heavy Chain Amino Acid Sequence
NO.
Light Chain Amino Acid Sequence
NO.

CD3B311
HC Peptide ID: CD3H218
(637)
LC Peptide ID: CD3L123
(645)

(BLW-2B4)
QVQLQQSGPGLVKPSQTLSLTCAISGDSVFN

QSALTQPASVSGSPGQSITISCTGTSSNIGT

NNAAWTWIRQSPSRGLEWLGRTYYRSKWLYD

YKFVSWYQQHPDKAPKVLLYEVSKRPSGVSS

YAVSVKSRITVNPDTSRNQFTLQLKSVTPED

RFSGSKSGNTASLTISGLQAEDQADYHCCSY

TALYYCSRGYSSSFDYWGQGTLVTVSSASTK

AGSGTLLFGGGTKLTVLGQPKAAPSVTLFPP

GPSVFPLAPSSKSTSGGTAALGCLVKDYFPE

SSEELQANKATLVCLISDFYPGAVTVAWKAD

PVTVSWNSGALTSGVHTFPAVLQSSGLYSLS

SSPVKAGVETTTPSKQSNNKYAASSYLSLTP

SVVTVPSSSLGTQTYICNVNHKPSNTKVDKK

EQWKSHRSYSCQVTHEGSTVEKTVAPTECS

VEPKSCDKTHTCPPCPAPELLGGPSVFLFPP

KPKDTLMISRTPEVTCVVVDVSHEDPEVKFN

WYVDGVEVHNAKTKPREEQYNSTYRVVSVLT

VLHQDWLNGKEYKCKVSNKALPAPIEKTISK

AKGQPREPQVYTLPPSREEMTKNQVSLTCLV

KGFYPSDIAVEWESNGQPENNYKTTPPVLDS

DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA

LHNHYTQKSLSLSPGK

CD3B312
HC Peptide ID: CD3H219
(640)
LC Peptide ID: CD3L124
(646)

(BLW-2E6)
QVRLQQSGPGLVKPSQTLSLTCAISGDSVFN

QSALTQPASVSGSPGQSITISCTGTSSNIGT

NNAAWSWIRQSPSRGLEWLGRTYYRSKWLYD

YKFVSWYQQHPDKAPKVLLYEVSKRPSGVSS

YAVTVKSRITVNPDTSRNQFTLQLTSVTPED

RFSGSKSGNTASLTISGLQAEDQADYHCCSY

TALYYCARGYSSSFDYWGQGTLVTVSSASTK

AGSGTLLFGGGTKLTVLGQPKAAPSVTLFPP

GPSVFPLAPSSKSTSGGTAALGCLVKDYFPE

SSEELQANKATLVCLISDFYPGAVTVAWKAD

PVTVSWNSGALTSGVHTFPAVLQSSGLYSLS

SSPVKAGVETTTPSKQSNNKYAASSYLSLTP

SVVTVPSSSLGTQTYICNVNHKPSNTKVDKK

EQWKSHRSYSCQVTHEGSTVEKTVAPTECS

VEPKSCDKTHTCPPCPAPELLGGPSVFLFPP

KPKDTLMISRTPEVTCVVVDVSHEDPEVKFN

WYVDGVEVHNAKTKPREEQYNSTYRVVSVLT

VLHQDWLNGKEYKCKVSNKALPAPIEKTISK

AKGQPREPQVYTLPPSREEMTKNQVSLTCLV

KGFYPSDIAVEWESNGQPENNYKTTPPVLDS

DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA

LHNHYTQKSLSLSPGK

CD3B313
HC Peptide ID: CD3H218
(638)
LC Peptide ID: CD3L125
(649)

(BLW-3B4)
QVQLQQSGPGLVKPSQTLSLTCAISGDSVFN

QSALTQPASVSGSPGQSITISCTGTSSNIGT

NNGAWSWIRQSPSRGLEWLGRTYYRSKWLYD

YKFVSWYQQHPDKAPKVLLYEVSKRPSGVSS

YAVSVKSRITVNPDTSRNQFTLQLNSVTPED

RFSGSKSGNTASLTISGLQAEDQADYHCCSY

TALYYCARGYSSSFDYWGQGTLVTVSSASTK

AGSGTLLFGGGTKLTVLGQPKAAPSVTLFPP

GPSVFPLAPSSKSTSGGTAALGCLVKDYFPE

SSEELQANKATLVCLISDFYPGAVTVAWKAD

PVTVSWNSGALTSGVHTFPAVLQSSGLYSLS

SSPVKAGVETTTPSKQSNNKYAASSYLSLTP

SVVTVPSSSLGTQTYICNVNHKPSNTKVDKK

EQWKSHRSYSCQVTHEGSTVEKTVAPTECS

VEPKSCDKTHTCPPCPAPELLGGPSVFLFPP

KPKDTLMISRTPEVTCVVVDVSHEDPEVKFN

WYVDGVEVHNAKTKPREEQYNSTYRVVSVLT

VLHQDWLNGKEYKCKVSNKALPAPIEKTISK

AKGQPREPQVYTLPPSREEMTKNQVSLTCLV

KGFYPSDIAVEWESNGQPENNYKTTPPVLDS

DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA

LHNHYTQKSLSLSPGK

CD3B314
HC Peptide ID: CD3H220
(641)
LC Peptide ID: CD3L126
(650)

(BLX-1F8)
QVQLQQSGPGLVKPSQTLSLTCAISGDSVFN

QSALTQPASVSGSPGQSITISCTGTSSDIGT

NNAAWSWIRQSPSRGLEWLGRTYYRSKWLYD

YKFVSWYQQHPDKAPKVLLYEVSKRPSGVSS

YAVSVKSRITVNPDTSRNQFTLQLKSVTPED

RFSGSKSDNTASLTISGLQAEDQADYHCCSY

TALYYCSRGYSSSFDYWGQGTLVTVSSASTK

AGSGTLLFGGGTKLTVLGQPKAAPSVTLFPP

GPSVFPLAPSSKSTSGGTAALGCLVKDYFPE

SSEELQANKATLVCLISDFYPGAVTVAWKAD

PVTVSWNSGALTSGVHTFPAVLQSSGLYSLS

SSPVKAGVETTTPSKQSNNKYAASSYLSLTP

SVVTVPSSSLGTQTYICNVNHKPSNTKVDKK

EQWKSHRSYSCQVTHEGSTVEKTVAPTECS

VEPKSCDKTHTCPPCPAPELLGGPSVFLFPP

KPKDTLMISRTPEVTCVVVDVSHEDPEVKFN

WYVDGVEVHNAKTKPREEQYNSTYRVVSVLT

VLHQDWLNGKEYKCKVSNKALPAPIEKTISK

AKGQPREPQVYTLPPSREEMTKNQVSLTCLV

KGFYPSDIAVEWESNGQPENNYKTTPPVLDS

DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA

LHNHYTQKSLSLSPGK

CD3B315
HC Peptide ID: CD3H221
(642)
LC Peptide ID: CD3L124
(647)

(BLX-2E9)
QVQLQQSGPGLVKPSQTLSLTCAISGDSVFN

QSALTQPASVSGSPGQSITISCTGTSRDIGT

NNAAWSWIRQSPSRGLEWLGRTYYRSKWLYD

YKFVSWYQQHPDKAPKVLLYEVSKRPSGVSS

YAVSVKSRITVNPDTSRNQFTLQLNSVTPED

RFSGSKSGNTASLTISGLQAEDQADYHCCSY

TALYYCVRGYSSSFDYWGQGTLVTVSSASTK

AGSGTLLFGGGTKLTVLGQPKAAPSVTLFPP

GPSVFPLAPSSKSTSGGTAALGCLVKDYFPE

SSEELQANKATLVCLISDFYPGAVTVAWKAD

PVTVSWNSGALTSGVHTFPAVLQSSGLYSLS

SSPVKAGVETTTPSKQSNNKYAASSYLSLTP

SVVTVPSSSLGTQTYICNVNHKPSNTKVDKK

EQWKSHRSYSCQVTHEGSTVEKTVAPTECS

VEPKSCDKTHTCPPCPAPELLGGPSVFLFPP

KPKDTLMISRTPEVTCVVVDVSHEDPEVKFN

WYVDGVEVHNAKTKPREEQYNSTYRVVSVLT

VLHQDWLNGKEYKCKVSNKALPAPIEKTISK

AKGQPREPQVYTLPPSREEMTKNQVSLTCLV

KGFYPSDIAVEWESNGQPENNYKTTPPVLDS

DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA

LHNHYTQKSLSLSPGK

CD3B316
HC Peptide ID: CD3H222
(643)
LC Peptide ID: CD3L124
(646)

(BLX-3F4)
QVQLQQSGPRLVRPSQTLSLTCAISGDSVFN

NNAAWSWIRQSPSRGLEWLGRTYYRSKWLYD

QSALTQPASVSGSPGQSITISCTGTSSNIGT

YAVSVKSRITVNPDTSRNQFTLQLNSVTPED

YKFVSWYQQHPDKAPKVLLYEVSKRPSGVSS

TALYYCARGYSSSFDYWGQGTLVTVSSASTK

RFSGSKSGNTASLTISGLQAEDQADYHCCSY

GPSVFPLAPSSKSTSGGTAALGCLVKDYFPE

AGSGTLLFGGGTKLTVLGQPKAAPSVTLFPP

PVTVSWNSGALTSGVHTFPAVLQSSGLYSLS

SSEELQANKATLVCLISDFYPGAVTVAWKAD

SVVTVPSSSLGTQTYICNVNHKPSNTKVDKK

SSPVKAGVETTTPSKQSNNKYAASSYLSLTP

VEPKSCDKTHTCPPCPAPELLGGPSVFLFPP

EQWKSHRSYSCQVTHEGSTVEKTVAPTECS

KPKDTLMISRTPEVTCVVVDVSHEDPEVKFN

WYVDGVEVHNAKTKPREEQYNSTYRVVSVLT

VLHQDWLNGKEYKCKVSNKALPAPIEKTISK

AKGQPREPQVYTLPPSREEMTKNQVSLTCLV

KGFYPSDIAVEWESNGQPENNYKTTPPVLDS

DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA

LHNHYTQKSLSLSPGK

CD3B317
HC Peptide ID: CD3H223
(644)
LC Peptide ID: CD3L124
(648)

(BLX-3G8)
QVQLQQSGPGLVKPSQTLSLTCAISGDSVFN

QSALTQPASVSGSPGQSITISCTGTSRDIGT

NNAAWSWIRQSPSRGLEWLGRTYYRSKWLYD

YKFVSWYQQHPDKAPKVLLYEVNKRPSGVSS

YAVSVKSRITVNPDTSRNQFTLQLNSVTPED

RFSGSKSGNTASLTISGLQAEDQADYHCCSY

TALYYCVRGYSSSFDYWGQGTLVTVSSASTK

AGSGTLLFGGGTKLTVLGQPKAAPSVTLFPP

GPSVFPLAPSSKSTSGGTAALGCLVKDYFPE

SSEELQANKATLVCLISDFYPGAVTVAWKAD

PVTVSWNSGALTSGVHTFPAVLQSSGLYSLS

SSPVKAGVETTTPSKQSNNKYAASSYLSLTP

SVVTVPSSSLGTQTYICNVNHKPSNTKVDKK

EQWKSHRSYSCQVTHEGSTVEKTVAPTECS

VEPKSCDKTHTCPPCPAPELLGGPSVFLFPP

KPKDTLMISRTPEVTCVVVDVSHEDPEVKFN

WYVDGVEVHNAKTKPREEQYNSTYRVVSVLT

VLHQDWLNGKEYKCKVSNKALPAPIEKTISK

AKGQPREPQVYTLPPSREEMTKNQVSLTCLV

KGFYPSDIAVEWESNGQPENNYKTTPPVLDS

DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA

LHNHYTQKSLSLSPGK

TABLE 7A

VH and VL sequences with HC and LC isotype of 7 monoclonal CD3 antibodies

from the first panel of 9 described above (see Table 3). All HC

isotypes were huIgG1_G1m(17). All LC isotypes were huLambda 2.

Fab ID
Peptide ID
VH (SEQ ID NO:)
Peptide ID
VL (SEQ ID NO:)

CD3B311
CD3H218
qvqlqqsgpglvkpsqtlslt
CD3L123
qsaltqpasvsgspggsitisct

caisgdsvfnnnaawswirqs

gtsrdigtykfvswyqqhpdkap

psrglewlgrtyyrskwlydy

kvllyevnkrpsgvssrfsgsks

avsvksritvnpdtsrnqftl

gntasltisglqaedqadyhccs

qlnsvtpedtalyycvrgyss

yagsgtllfgggtkltvl

sfdywgqgtlvtvss

(658)

(651)

CD3B312
CD3H219
qvqlqqsgprlvrpsqtlslt
CD3L124
qsaltqpasvsgspggsitisct

caisgdsvfnnnaawswirqs

gtssnigtykfvswyqqhpdkap

psrglewlgrtyyrskwlydy

kvllyevskrpsgvssrfsgsks

avsvksritvnpdtsrnqftl

gntasltisglqaedqadyhccs

qlnsvtpedtalyycargyss

yagsgtllfgggtkltvl

sfdywgqgtlvtvss

(659)

(652)

CD3B313
CD3H218
qvqlqqsgpglvkpsqtlslt
CD3L124
qsaltqpasvsgspggsitisct

caisgdsvfnnnaawswirqs

gtsrdigtykfvswyqqhpdkap

psrglewlgrtyyrskwlydy

kvllyevskrpsgvssrfsgsks

avsvksritvnpdtsrnqftl

gntasltisglqaedqadyhccs

qlnsvtpedtalyycvrgyss

yagsgtllfgggtkltvl

sfdywgqgtlvtvss

(688)

(687)

CD3B314
CD3H220
qvqlqqsgpglvkpsqtlslt
CD3L126
qsaltqpasvsgspggsitisct

caisgdsvfnnnaawswirqs

gtssdigtykfvswyqqhpdkap

psrglewlgrtyyrskwlydy

kvllyevskrpsgvssrfsgsks

avsvksritvnpdtsrnqftl

dntasltisglqaedqadyhccs

qlksvtpedtalyycsrgyss

yagsgtllfgggtkltvl

sfdywgqgtlvtvss

(660)

(653)

CD3B315
CD3H221
qvqlqqsgpglvkpsqtlslt
CD3L124
qsaltqpasvsgspggsitisct

caisgdsvfnnngawswirqs

gtssnigtykfvswyqqhpdkap

psrglewlgrtyyrskwlydy

kvllyevskrpsgvssrfsgsks

avsvksritvnpdtsrnqftl

gntasltisglqaedqadyhccs

qlnsvtpedtalyycargyss

yagsgtllfgggtkltvl

sfdywgqgtlvtvss

(659)

(654)

CD3B316
CD3H222
qvrlqqsgpglvkpsqtlslt
CD3L124
qsaltqpasvsgspggsitisct

caisgdsvfnnnaawswirqs

gtssnigtykfvswyqqhpdkap

psrglewlgrtyyrskwlydy

kvllyevskrpsgvssrfsgsks

avtvksritvnpdtsrnqftl

gntasltisglqaedqadyhccs

qltsvtpedtalyycargyss

yagsgtllfgggtkltvl

sfdywgqgtlvtvss

(659)

(655)

CD3B317
CD3H223
qvqlqqsgpglvkpsqtlslt
CD3L124
qsaltqpasvsgspggsitisct

caisgdsvfnnnaawtwirqs

gtssnigtykfvswyqqhpdkap

psrglewlgrtyyrskwlydy

kvllyevskrpsgvssrfsgsks

avsvksritvnpdtsrnqftl

gntasltisglqaedqadyhccs

qlksvtpedtalyycsrgyss

yagsgtllfgggtkltvl

sfdywgqgtlvtvss

(659)

(656)

TABLE 7B

CDR sequences with HC and LC isotype of 7 monoclonal CD3

antibodies from the first panel of 9 described above

(see Table 3). Sequences are defined according

to Kabat. All HC isotypes were huIgG1_G1m(17).

All LC isotypes were huLambda 2.

CDRs (SEQ ID NO:)

FAB ID
Peptide ID
CDR1
CDR2
CDR3

CB3B311
HC
NNNAAWS
RTYYRSKWLYDYAVSVKS
GYSSSFDY

CD3H218
(662)
(663)
(664)

LC
TGTSRDIGTYKFVS
EVNKRPS
CSYAGSGTLL

CD3L123
(667)
(669)
(670)

CD3B312
HC
NNNAAWS
RTYYRSKWLYDYAVSVKS
GYSSSFDY

CD3H219
(662)
(663)
(664)

LC
TGTSSNIGTYKFVS
EVSKRPS
CSYAGSGTLL

CD3L124
(671)
(673)
(670)

CD3B313
HC
NNNAAWS
RTYYRSKWLYDYAVSVKS
GYSSSFDY

CD3H218
(662)
(663)
(664)

LC
TGTSRDIGTYKFVS
EVSKRPS
CSYAGSGTLL

CD3L124
(672)
(673)
(670)

CD3B314
HC
NNNAAWS
RTYYRSKWLYDYAVSVKS
GYSSSFDY

CD3H220
(662)
(663)
(664)

LC
TGTSSDIGTYKFVS
EVSKRPS
CSYAGSGTLL

CD3L126
(674)
(673)
(670)

CD3B315
HC
NNNGAWS
RTYYRSKWLYDYAVSVKS
GYSSSFDY

CD3H221
(665)
(663)
(664)

LC
TGTSSNIGTYKFVS
EVSKRPS
CSYAGSGTLL

CD3L124
(671)
(673)
(670)

CD3B316
HC
NNNAAWS
RTYYRSKWLYDYAVTVKS
GYSSSFDY

CD3H222
(662)
(689)
(664)

LC
TGTSSNIGTYKFVS
EVSKRPS
CSYAGSGTLL

CD3L124
(671)
(673)
(670)

CD3B317
HC
NNNAAWT
RTYYRSKWLYDYAVSVKS
GYSSSFDY

CD3H223
(666)
(663)
(664)

LC
TGTSSNIGTYKFVS
EVSKRPS
CSYAGSGTLL

CD3L124
(671)
(673)
(670)

1-3 Screening of Hybridomas for Binding to Human and Cynomolgus Purified T Cells

A cell-based binding assay was designed to assess the binding capacity of individual rat hybridoma supernatants to human (FIG. 2) and cynomolgus (FIG. 3) purified CD3+T lymphocytes. The T cells were counted, diluted to 1×10{circumflex over ( )}6 cells/mL and incubated with 0.5 ul/mL of Live/Dead Fixable Green Dead Cell Stain (Life Technologies, L-2301). Next the cells were aliquoted into a U-bottom (Falcon 353077) plate at 100 ul/well (1×10{circumflex over ( )}5 cells/well). The plates were centrifuged at 300 g for 5 minutes to pellet the cells and the supernatant removed. The plate was vortexed briefly to resuspend the cells. The hybridoma supernatants were diluted in FACS staining buffer (BSA, BD Biosciences 554657) to 4.5 μg/mL and then serially diluted 6 times at 1:3 dilutions to the lowest concentration of 0.006 μg/mL. A positive control of mouse anti-human CD3 (SP34-2, BD Biosciences 551916) and negative isotype control (mouse IgG1 BD Biosciences 556648) were also diluted to 4.5 μg/mL. 50 μl of each sample was added to the T cells and incubated at 4° C. for 1 hour. The cells were washed once with staining buffer and secondary AF647 Goat anti-mouse IgG (Life Technologies, A21235) or AF647 goat anti-rat IgG (Life Technologies, A21247) was added at 10 μg/mL in 50 μl was added according to the appropriate species (anti-rat for hybridoma samples and anti-mouse for the control antibodies). The plates were incubated at 4° C. for 45 minutes and washed with staining buffer twice. The cells were resuspended in 25 μl of running buffer (staining buffer+1 mM EDTA (Life technologies, AM9260G)+0.1% pluronic F-68 (Life Technologies 24040-032)) and read on the Intellicyt system (Intellicyt Corp.). The results are shown in FIGS. 2 and 3.

In other experiments, purified human T cells were plated at 1.1×10{circumflex over ( )}5 cells/well in U-bottom plates. The plates were centrifuged at 300 g for 5 minutes to pellet the cells and the supernatant removed. The plate was vortexed briefly to resuspend the cells. The hybridoma supernatants were diluted in FACS staining buffer (BSA, BD Biosciences 554657) to 30 ug/mL and then serially diluted 11 times at 1:3 dilutions to the lowest concentration of 0.00017 ug/mL. 50 u of each sample was added to the T cells and incubated at 4 C for 1 hour. The cells were washed once with staining buffer and secondary Dylight 650 goat anti-rat IgG (Bethyl, A110-239D5) was added at 10 ug/mL in 50 u was added. The plates were incubated at 4 C for 1 hour and washed with staining buffer twice. The cells were resuspended in 30 u of FACS buffer and read on the Hypercyte flow cytometer (Intellicyt Corp.). Representative dose-response curves for the anti-CD3 clones binding to primary human T cells are shown in FIG. 5.

Representative competition binding curves with SP34-2 (a commercial anti-human CD3 antibody which has a known epitope and is cross-reactive with cynomolgus CD3) are shown in FIG. 6. The initial screening results are summarized in Table 8. Six of the clones showed positive binding and also compete with SP34-2 for binding to primary human T cells.

1-4 Competition Assay with SP34-2 Commercial CD3 Antibody

The hybridoma supernatants were also assessed for their ability to compete with commercial anti-human CD3 antibody SP34-2 which has a known epitope and is cross-reactive with cynomolgus CD3. First a concentration titration curve of AF488 fluorescently labelled SP34-2 (BD, 557705) was performed to determine a fixed concentration of SP34-2 for the following competition assays. Briefly, human purified T cells were diluted to 1×10{circumflex over ( )}6 cell/mL in PBS. Fc block (Human TruStain Fc Block, Biolegend, 422302) was added at 5 μL/100 μL cells and plated at 100 μL/well in U-bottom plates. AF488 SP34-2 and the AF488 labelled isotype control (AF488 mouse IgG1, BD, 400129) were serially diluted from 50 μg/mL to 0.049 μg/mL in a 1:2 dilution scheme. The plates were centrifuged at 300×g for 5 minutes to pellet the cells and the supernatant removed. The plate was gently vortexed briefly to resuspend the cells. 504 of each AF488 SP34-2 dilution was added to the cells and incubated at 4 C for 1 hour. The plates were washed with staining buffer twice and once with running buffer (staining buffer+1 mM EDTA (Life technologies, AM9260G)+0.1% pluronic F-68 (Life Technologies 24040-032)). The cells were resuspended in 254 of running buffer and read on the HTFC Screening System (IntelliCyt Corporation). Based on the dose response curve, a fixed concentration of 2 ug/mL SP34-2 was chosen for the competition assay.

Seven hybridomas that demonstrated binding to purified T cells were assayed for competition with SP34-2 binding to human T cells (FIG. 4). Control antibodies were included in the competition assay. Unlabeled Mouse Anti-Human CD3, SP34-2 antibody and Mouse Anti-Human CD3, UCHT1 antibody were used as positive controls and rat IgG and Mouse isotype were used as negative controls for AF488 labeled SP34-2. Purified human T cells were diluted to a concentration of 1×10{circumflex over ( )}6 cells/mL in PBS. Fc block at 54 per 100 μL cells (Human TruStain Fc Block, Biolegend, 422302) and Live/Dead Fixable Far Red Dead Cell Stain at 0.54 per mL cells (Life Technologies L10120) were added to the cells and incubated for 15 minutes at 4° C. Next, 10⁵cells per well (1×10{circumflex over ( )}5 cells/well) were aliquoted into a 96-well U-bottom plate (Falcon 353077). The plates were centrifuged at 300×g for 5 minutes to pellet the cells and the supernatant removed. The plate was gently vortexed briefly to resuspend the cells. The hybridoma supernatants and the control antibodies were diluted in FACS staining buffer (BSA, BD Biosciences 554657) at 2× the desired final concentration. 354 of the 2× hybridoma supernatants and the control antibodies were mixed with 35 uL of 2×AF488 SP34-2 (4 μg/mL) to give the desired 1× concentration of hybridoma supernatants, 1× concentration of control antibodies and 2 μg/mL AF488 SP34-2. The hybridoma supernatants and control antibodies were assayed using a 7-point titration with a concentration range. Hybridoma supernatants were assayed from 200 μg/mL to 0.08 μg/mL and control antibodies were assayed from 100 μg/mL to 0.04 μg/mL. 50 uL of the 1× hybridoma supernatants or 1× control antibodies with 2 μg/mL AF488 SP34-2 were added to the T cells and incubated at 4° C. for 2 hours. The plates were washed with staining buffer twice and once with running buffer (staining buffer+1 mM EDTA (Life technologies, AM9260G)+0.1% pluronic F-68 (Life Technologies 24040-032)). The cells were resuspended in 254 of running buffer and read on the HTFC Screening System (IntelliCyt Corporation). Representative competition binding curves with SP34-2 (a commercial anti-human CD3 antibody which has a known epitope and is cross-reactive with cynomolgus CD3) are shown in FIG. 4 and FIG. 6. The initial screening results are summarized in Table 8. Six of the clones showed positive binding and also competed with SP34-2 for binding to primary human T cells.

As shown in FIG. 4 the seven antibodies competed with SP34-2 with similar curves. The right shift of the curves relative to the control SP34-2 indicates a weaker binding affinity. The isotype control rat IgG did not compete with SP34-2, as expected.

TABLE 8

Summary of anti-CD3 antibody binding on primary human

T cells. Anti-CD3 clones BLX-4E5, BLX-5H7, BLX-8B4,

BLX-8B6, BLX-8G8 and BLW-1E3 were positive for binding

to human T cells and competed with SP34-2 binding

Ab clone
Binds with human T cells
Competes with SP34-2

BLW-2B4
+
+

BLW-2E6
+
+

BLW-3B4
+
+

BLX-1F8
+
+

BLX-2E9
+
+

BLX-3F4
+
+

BLX-3G8
+
+

BLX-1G10
−
−

BLX-3H6
−
−

BLX-4E5
+
+

BLX-5H7
+
+

BLX-8B4
+
+

BLX-8B6
+
+

BLX-8G8
+
+

BLW-1E3
+
+

BLW-1F1
+
−

BLW-2C4
−
−

BLW-2C11
−
−

BLW-2F9
−
−

BLW-3B5
−
−

BLW-3H5
−
−

1-5 Screening of Hybridoma Hits for T Cell Activation as Measured by CD69 Upregulation

A primary human and cynomolgus monkey T cell based assay was used to determine the capacity of the hybridoma hits to activate T cells. This was accomplished by coating antibodies to a plate to mimic the cross-linking effect of TCR activation. Upon activation, T cells are known to upregulate the surface expression of the protein, CD69. The experiment was conducted by coating 50 μl of a 10 μg/ml antibody preparation with unknown samples or controls (positive control: in house, Okt-3 BISB264.002, BD Bioscience SP-34-2 #551916; negative control: anti-CD20 in house BISB266.004) to a 96 well plate (Costar #3361). The plates were incubated overnight at 4° C. The next day the plates were washed twice with PBS. Frozen primary T cells (human sourced from Biological Specialities or Hemacare; cynomolgus sourced from WorldWide Primates) were thawed, counted for viability and resuspended at 2×10⁶cell/ml in RPMI 1640 medium (Gibco #11875 with 10% HI FBS (Gibco #10062). 100 μl of the cells was added to the plate and incubated overnight (approximately 16 hours) at 37 C, 5% CO₂. The following day the plates were spun at 1300 rpm for 3 minutes to pellet the cells and the supernatants were discarded. The cells were washed once in PBS and spun as before. 10 μl of a 2.5% solution of Live/Dead Green Fixable Dye (Life Technologies #L23101) in PBS was added to each well and incubated at room temperature and in the dark for 10 minutes. Next 50 μl of a 1% solution of anti-CD69 AF488 (Biolegend #310916 lot #B125271) in FACS buffer (BD Biosciences #554657) was added and the plates were incubated for 45 minutes at 4° C. The plates were washed twice by pelleting the cells as before and discarding the supernatant and resuspending in 150 μl of FACS buffer. After the final wash the cells were resuspended in 150 μl of FACS buffer and read on the FACS Canto. As seen in FIG. 20, the positive controls cOkt3 and SP34-2 induced the upregulation of CD69 on human T cells as indicated by the measured mean fluorescence intensity of the anti-CD69 staining. Only SP34-2 induced CD69 expression in the cynomolgus T cells, as it binds to a region of CD3 sequence that is conserved from monkey to man. The OKT3 anti-CD3 clone did not bind cynomolgus CD3 and did not induce CD69 upregulation. The negative control in both human and cynomolgus T cells was an anti-CD20 which is not expressed on T cells. Of the hybridoma clones that were tested for T cell activation, several induced CD69 expression to a similar degree as the positive control, namely 2B4, 2E6, 3B4, 1F8, 2E9, 3F4, 3G8, 4E5, 5H7, 8B4, 8G8 and 1F1. Most also bound and activated cynomolgus T cells except for 5H7, 8B4, and 8G8.

1-6 Framework Engineering of BLW-2E6

Clones showed high homology and carried framework mutations comparing to human immunoglobulin germline sequences (FIGS. 1A and 1B). Clone 2E6 was selected to adapt the standard framework sequence. All 6 mutations on HC and 7 mutations on LC were mutated back to human germline sequence, either individually or combined (Table 9). The mutated V-region DNA sequences were synthesized and cloned into the same mammalian expression vectors as their parental constructs. The HC and LC constructs were paired via matrix format to generate proteins carrying individual or combinatorial mutations and protein activity was tested. V48 on LC could not be changed back to germline. All other back mutations were not critical but reduced activity to some degree.

TABLE 9

BLW-2E6 framework variants.

peptide ID

Heavy chain back mutation to human germline

WT
CD3H219

R10G
CD3H225

R13K
CD3H226

V73I
CD3H227

R79K
CD3H228

T83S
CD3H229

L96V
CD3H230

R10G/R13K/V73I/R79K/T83S/L96V
CD3H231

Light chain back mutation to human germline

WT
CD3L124

D43G
CD3L128

V48L
CD3L129

L49M
CD3L130

L50I
CD3L131

S62N
CD3L132

Q85E
CD3L133

H89Y
CD3L134

D43G/L49M/L50I/S62N/Q85E
CD3L135

D43G/L49M/L50I/S62N/Q85E/H89Y
CD3L137

D43G/V48L/L49M/L50I/S62N/Q85E/H89Y
CD3L136

D43G/L49M/L50I/S62N/Q85E/H89Y/C91V
CD3L197

Framework mutations were engineered to one hybridoma clone, BLW-2E6, resulting in 80 mutant clones, some of which are indicated in Table 10. The 80 mutant clones were assayed for binding to primary human T cells (FIGS. 7 and 8). The T cells were counted, diluted to 1×10{circumflex over ( )}6 cells/mL and incubated with 54 Fc block (Human TruStain Fc Block, Biolegend, 422302) per 100 μL cells and 0.5 μl per mL of Live/Dead Fixable Green Dead Cell Stain (Life Technologies, L-2301) per 100 μL cells. Next, the cells were aliquoted into a 96-well U-bottom plate (Falcon 353077) at 100 μL/well (1×10{circumflex over ( )}5 cells/well). The plates were centrifuged at 300×g for 5 minutes to pellet the cells and the supernatant removed. The plate was gently vortexed briefly to resuspend the cells. The hybridoma supernatants were diluted in FACS staining buffer (BSA, BD Biosciences 554657) to 7.5 μg/mL, 1.5 ug/mL, 0.3 μg/mL, and 0.06 μg/mL. 504 of each sample was added to the T cells and incubated at 4° C. for 1 hour. The cells were washed once with staining buffer, and 504 of 5 μg/mL secondary AF647 goat anti-Human IgG F(ab′)2 (Jackson ImmunoResearch catalog 109-605-097) was added to the cells. The plates were incubated at 4° C. for 45 minutes and washed with staining buffer twice and once with running buffer (staining buffer+1 mM EDTA (Life technologies, AM9260G)+0.1% pluronic F-68 (Life Technologies 24040-032)). The cells were resuspended in 254 of running buffer and read on the HTFC Screening System (IntelliCyt Corporation). The results demonstrate that a change in the LC at position 48 abolished binding so that mutation was not carried forward. A slight reduction in binding was seen in the HC with all positions returned to germline (CD3H231).

1-7 C91 Scanning of BLW-2E6 LC

Clone 2E6 and its derivatives expressed poorly and protein aggregation was observed. One residue, C91 of light chain, was predicted to have post-translational modification (PTM) risk and was mutated to all other possible amino acids to improve protein stability (Table 10). The mutated V-region DNA sequences were synthesized and cloned into the same human lambda expression vector as their parental construct. The SPR results demonstrated a change to valine or leucine at position 91 did not dramatically alter binding affinity. This change was also incorporated into wild type sequence and the framework adaptation above resulting in antibodies CD3B376 (CD3H219/CD3L150) and CD3B450 (CD3H231/CD3L197). CD3B376 and CD3B450 were cloned as IgG4PAA (IgG4 with S228P, F234A, L235A mutations).

Sequence information for CD3B376 is provided below:

CD3H219 HC amino acid sequence (SEQ ID NO: 640):

QVQLQQSGPRLVRPSQTLSLTCAISGDSVFNNNAAWSWIRQSPSRGLEWLGRTYYRSKWLYDYAVSVKSRITVN

PDTSRNQFTLQLNSVTPEDTALYYCARGYSSSFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCL

VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKY

GPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQF

NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVK

GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFLLYSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLS

LGK

CD3H219 HC nucleic acid sequence (SEQ ID NO: 712)

caggtgcagctgcagcagtctggccctagactcgtgcggccttcccagaccctgtctctgacctgtgccatctc

cggcgactccgtgttcaacaacaacgccgcctggtcctggatccggcagagcccttctagaggcctggaatggc

tgggccggacctactaccggtccaagtggctgtacgactacgccgtgtccgtgaagtcccggatcaccgtgaac

cctgacacctcccggaaccagttcaccctgcagctgaactccgtgacccctgaggacaccgccctgtactactg

cgccagaggctactcctcctccttcgactattggggccagggcaccctcgtgaccgtgtcctct

CD3L150 LC amino acid sequence (SEQ ID NO: 676):

QSALTQPASVSGSPGQSITISCTGTSSNIGTYKFVSWYQQHPDKAPKVLLYEVSKRPSGVSSRFSGSKSGNTAS

LTISGLQAEDQADYHCVSYAGSGTLLFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAV

TVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

CD3L150 LC nucleic acid sequence (SEQ ID NO: 713)

cagtctgctctgacccagcctgcctccgtgtctggctctcccggccagtccatcaccatcagctgtaccggcac

ctcctccaacatcggcacctacaagttcgtgtcctggtatcagcagcaccccgacaaggcccccaaagtgctgc

tgtacgaggtgtccaagcggccctctggcgtgtcctccagattctccggctccaagtctggcaacaccgcctcc

ctgaccatcagcggactgcaggctgaggaccaggccgactaccactgtgtgtcctacgctggctctggcaccct

gctgtttggcggaggcaccaagctgaccgtgctg

VH amino acid sequence of CD3H219 (SEQ ID NO: 652):

qvqlqqsgprlvrpsqtlsltcaisgdsvfnnnaawswirqspsrglewlgrtyyrskwlydyavsvksritvn

pdtsrnqftlqlnsvtpedtalyycargysssfdywgqgtlvtvss

VL amino acid sequence of CD3L150 (SEQ ID NO: 661):

qsaltqpasvsgspgqsitisctgtssnigtykfvswyqqhpdkapkvllyevskrpsgvssrfsgsksgntas

ltisglqaedqadyhcVsyagsgtllfgggtkltvl

HCDR1 of CD3H219 (SEQ ID NO: 662): NNNAAWS

HCDR2 of CD3H219 (SEQ ID NO: 663): RTYYRSKWLYDYAVSVKS

HCDR3 of CD3H219 (SEQ ID NO: 664): GYSSSFDY

LCDR1 of CD3L150 (SEQ ID NO: 671): TGTSSNIGTYKFVS

LCDR2 of CD3L150 (SEQ ID NO: 673): EVSKRPS

LCDR3 of CD3L150 (SEQ ID NO: 690): VSYAGSGTLL

Sequence information for CD3B450 is provided below:

CD3H231 HC amino acid sequence (SEQ ID NO: 675):

QVQLQQSGPGLVKPSQTLSLTCAISGDSVFNNNAAWSWIRQSPSRGLEWLGRTYYRSKWLYDYAVSVKSRITIN

PDTSKNQFSLQLNSVTPEDTAVYYCARGYSSSFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCL

VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKY

GPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQF

NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVK

GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFLLYSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLS

LGK

CD3H231 HC nucleic acid sequence (SEQ ID NO: 714):

caggtgcagctgcagcagagcggccccggcctggtcaagcccagccagaccctgagcctgacctgcgccatcag

cggcgacagcgtgttcaacaacaacgccgcctggtcctggatccgccagagccccagccgcggcctggagtggc

tgggccgcacctactaccgcagcaagtggctgtacgactacgccgtgtccgtgaagtcccgcatcaccatcaac

cccgacaccagcaagaaccagttctccctgcagctgaacagcgtgacccccgaggacaccgccgtgtactactg

cgcccgcggctacagcagcagcttcgactactggggccagggcaccctggtcaccgtgtccagc

CD3L197 LC amino acid sequence (SEQ ID NO: 677):

QSALTQPASVSGSPGQSITISCTGTSSNIGTYKFVSWYQQHPGKAPKVMIYEVSKRPSGVSNRFSGSKSGNTAS

LTISGLQAEDEADYYCVSYAGSGTLLFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAV

TVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

CD3L197 LC nucleic acid sequence (SEQ ID NO: 715):

Cagtctgctctgacccagcctgcctccgtgtctggctctcccggccagtccatcaccatcagctgtaccggcac

ctcctccaacatcggcacctacaagttcgtgtcctggtatcagcagcaccccggcaaggcccccaaagtgatga

tctacgaggtgtccaagcggccctccggcgtgtccaacagattctccggctccaagtccggcaacaccgcctcc

ctgacaatcagcggactgcaggccgaggacgaggccgactactactgtgtgtcctacgccggctctggcaccct

gctgtttggcggcggaacaaagctgaccgtgctg

VH amino acid sequence of CD3H231 (SEQ ID NO: 657):

qvqlqqsgpglvkpsqtlsltcaisgdsvfnnnaawswirqspsrglewlgrtyyrskwlydyavsvksritin

pdtsknqfslqlnsvtpedtavyycargysssfdywgqgtlvtvss

VL amino acid sequence of CD3L197 (SEQ ID NO: 678):

QSALTQPASVSGSPGQSITISCTGTSSNIGTYKFVSWYQQHPGKAPKVMIYEVSKRPSGVSNRFSGSKSGNTAS

LTISGLQAEDEADYYCVSYAGSGTLLFGGGTKLTVL

HCDR1 of CD3H231 (SEQ ID NO: 662): NNNAAWS

HCDR2 of CD3H231 (SEQ ID NO: 663): RTYYRSKWLYDYAVSVKS

HCDR3 of CD3H231 (SEQ ID NO: 664): GYSSSFDY

LCDR1 of CD3L197 (SEQ ID NO: 671): TGTSSNIGTYKFVS

LCDR2 of CD3L197 (SEQ ID NO: 673): EVSKRPS

LCDR3 of CD3L197 (SEQ ID NO: 690): VSYAGSGTLL

TABLE 10

Engineered variants of BLW-2E6 LC by C91 scanning

C91 scanning
peptide ID

CD3 2E6 LC
CD3L124

CD3 2E6 LC, C91S
CD3L146

CD3 2E6 LC, C91G
CD3L147

CD3 2E6 LC, C91E
CD3L148

CD3 2E6 LC, C91D
CD3L149

CD3 2E6 LC, C91V
CD3L150

CD3 2E6 LC, C91A
CD3L151

CD3 2E6 LC, C91R
CD3L152

CD3 2E6 LC, C91K
CD3L153

CD3 2E6 LC, C91N
CD3L154

CD3 2E6 LC, C91M
CD3L155

CD3 2E6 LC, C91I
CD3L156

CD3 2E6 LC, C91T
CD3L157

CD3 2E6 LC, C91W
CD3L158

CD3 2E6 LC, C91Y
CD3L159

CD3 2E6 LC, C91L
CD3L160

CD3 2E6 LC, C91F
CD3L161

CD3 2E6 LC, C91Q
CD3L162

CD3 2E6 LC, C91H
CD3L163

CD3 2E6 LC, C91P
CD3L164

1-8 Binding of BLW-2E6 CD3 mAbs to hCD3E Construct by ProteOn SPR

The binding of BLW-2E6 anti-CD3 mAbs with point mutations on the light and/or heavy chain(s) to a recombinant human CD3ε (1-27) peptide with a C-terminal Tencon25 fusion (Janssen production, referred as hCD3ε(1-27)-Tn25) was measured by ProteOn SPR (Bio-Rad). Goat anti-human Fc IgG (Jackson Immunoresearch, Cat #109-005-098) was directly immobilized via amine coupling at 30 μg/mL in acetate buffer, pH 5.0 on all 6 ligand channels in vertical orientation on GLC Sensor Chip (Bio-Rad, catalog no. 176-5011) with a flow rate of 30 μL/min in PBS containing 0.005% Tween-20. The immobilization densities averaged about 6000 Response Units (RU) with less than 5% variation among different channels. Five different mAbs were captured on the anti-human Fc IgG surface at 1.5 ug/ml (1000˜1250 RU) in vertical ligand orientation, with the 6th ligand channel as no ligand surface control. The hCD3ε(1-27)-Tn25 at 1 μM concentration in 3-fold dilution series of 5 concentrations flew in as analyte to bind to captured mAbs in the horizontal orientation. A 6th buffer sample was also injected to monitor the dissociation of captured mAb and baseline stability. The dissociation phase for all concentrations of hCD3ε(1-27)-Tn25 was monitored at a flow rate of 100 μL/min for 30 minutes. The binding surface was regenerated for the next interaction cycle using an 18 second pulse of 0.8% phosphoric acid to remove the antigen and the bound mAb. The raw data were processed by subtracting two sets of reference data from the response data: 1) the inter-spot signals to correct for the non-specific interactions between the Ag and the empty chip surface; 2) the buffer channel signals to correct for baseline drifting due to the dissociation of captured mAb surface over time. The processed data at all concentrations for each mAb were globally fit to a 1:1 simple Langmuir binding model to extract estimates of the kinetic (k_on, k_off) and affinity (K_D) constants. Results are provided in FIG. 11.

TABLE 11

Summary of the kinetics/affinities of BLW-2E6 variants binding to hCD3ε(1-27)-Tn25 (N = 1)

Protein
Heavy
Light
ka
kd
KD

AA ID
Chain
Chain
(1/Ms)
(1/s)
(nM)
HC Mutation(s)
LC mutation(s)

C3B312
CD3H219
CD3L124
9.08E+04
1.87E−03
20.6
Wild type
Wild type

CD3B376
CD3H219
CD3L150
1.02E+05
2.37E−03
23.3
Wild type
C91V

N/A
CD3H219
CD3L160
9.84E+04
4.05E−03
41.1
Wild type
C91L

N/A
CD3H219
CD3L137
6.13E+04
2.02E−03
32.9
Wild type
D43G/L49M/L50I/

S62N/Q85E/H89Y

CD3B334
CD3H231
CD3L137
6.96E+04
5.54E−03
79.6
R10G/R13K/V73I/
D43G/L49M/L50I/

R79K/T83S/L96V
S62N/Q85E/H89Y

N/A = not assigned

1-9 Binding of Anti-CD3 Monoclonal Antibodies to Human T Cells

In vitro binding affinity of CD3B376 and CD3B450 to human T cells was determined by flow cytometry after crossing with antigen specific target arm. A preliminary study was carried out on human T cells to determine the saturation binding constant of an anti-CD3 tracer molecule (KdT). A fixed concentration of the tracer ([T]) was then used in a competition binding assay with titrated concentrations of the test mAbs. The IC50 (concentration at which 50% inhibition is achieved) value of the test molecule was used to determine the binding affinity (K_d) using the following formula: K_d=IC50/(1+([T]/K_dT)). Five human donors were used to determine the saturation binding constant (K_dT) of the tracer, a commercially available AlexaFluor488 SP34-2 anti-CD3 (BioScience #557705) (data not shown).

Determining Saturation Binding Constant of Tracer (K_dT)

Methods: Human pan T cells were cryogenically-stored in nitrogen tanks until used. T cells were thawed, washed with PBS, re-suspended in FACS Staining buffer, counted (with viability noted), and re-suspended at 0.5×10{circumflex over ( )}6 cells/mL. Far Red Live/Dead stain (Life Technologies, AKA Invitrogen #L34974) (50 μL of DMSO into a vial) was added at 1 μL per 1×10{circumflex over ( )}6 cells; and FcR blocker (Miltenyi Biotec #130-059-901) (1 mL of a 1:20 dilution per 0.5×10{circumflex over ( )}6 cells) were added to the cells for 10 minutes each. Cells were plated at 50,000 cells/well and washed. Increasing concentrations of the AlexaFluor488 SP-34 anti-CD3 were added to the T cells for 2 hours at 4° C. Cells were washed to remove un-bound antibody, fixed for 15 minutes, washed, and re-suspended in FACS Staining buffer containing 1 mM EDTA.

The iQue Intellicyte Flow Cytometer was used to measure binding. Cells were gated for T cell population, followed by cell singlets, followed by Live Cells (FL4). Geometric mean of staining (FL1) was determined for each well.

The acquired mean fluorescence intensity values were plotted as a function of the antibody molecule concentration and analyzed using Prism software in a one-site binding analysis (Total Binding) (FIG. 9). The software calculates the corresponding K_dvalue that describes the binding of the antibody molecule to a receptor (the CD3 on Human Pan T cells) that follows the law of mass action. The formula is as follows: Y=(B_max×X)/(K_d+X); where: Bmax is maximal binding; K_dis the concentration of ligand required to reach half-maximal binding.

Results: K_dvalues were derived for each donor, and the mean value obtained. The Saturation Binding Constant (K_dT) for human T-Cells was derived to be 5.6±1.0 nM (n=4) and was used in the previously mentioned formula to determine K_dbinding affinities.

Determining Binding Affinity of Anti-CD3 mAbs by Competition Assay

Methods: Competition binding studies were performed using bivalent antibodies against CD3:

- Anti CD3 bivalents: CD3B376 and CD3B450 (FIG. 10) Human pan T cells were used to determine the binding affinity of the test mAbs. The tracer used was the commercially available AlexaFluor488 SP-34 anti-CD3 (BioScience #557705) and the saturation binding constant for this tracer is described above.

T cells were cryogenically-stored in nitrogen tanks until used. T cells were thawed, washed with PBS, re-suspended in FACS Staining buffer, counted with viability noted, and re-suspended at 0.5×10⁶cells/mL. Far Red Live/Dead stain (Life Technologies, AKA Invitrogen #L34974) (50 μL of DMSO into a vial) was added at 1 μL per 1×10{circumflex over ( )}6 cells; and FcR blocker (Miltenyi Biotec #130-059-901) (1 mL of a 1:20 dilution per 0.5×10{circumflex over ( )}6 cells) was added to the cells for 10 minutes each. Cells were plated at 50,000 cells/well and washed.

The mAbs (and isotype control), were serially diluted 1:2 from a starting concentration of 1000 or 200 μg/mL (2×), and a fixed concentration of the tracer (5 μg/mL; 2×) was mixed together to give 1× concentrations. Therefore, the final (1×) concentration of the tracer was 2.5 μg/mL=16.6 nM. The mixture was added to the T cells for 2 hours at 4° C. Cells were then washed to remove un-bound antibody, fixed for 15 minutes, washed, and re-suspended in FACS Staining buffer containing 1 mM EDTA.

The iQue Intellicyte Flow Cytometer was used to measure binding. Cells were gated for T-cell population, followed by cell singlets, followed by Live Cells (FL4). Geometric mean of staining (FL1) was determined for each well. The acquired mean fluorescence intensity values were plotted as a function of the log antibody molecule concentration (converted to nM) and analyzed using Prism software in a sigmoidal dose-response (variable slope) from which the EC50/IC50 values (in nM) are derived. The binding affinity (K_d) was derived using the following formula: K_d=IC50/(1+([T]/K_dT)). Where: K_dis the affinity of the competitor (unlabeled molecule); IC50 in nM of the test compound; [T] is concentration of the tracer (16.6 nM); K_dT is the K_dof the tracer determined by saturation binding (5.6 nM for human).

Production of Monoclonal and Bispecific Antibodies

The bispecific CD3 antibodies of the present invention may be generated through controlled Fab-arm exchanged (FAE) as described in Labrijn et al., 2013, PNAS, vol. 110(13): 5145-5150; PCT Publ. WO 2011/131746; or in Labrijn et al., 2014, Nat Protoc, 9(10):2450-63. Briefly, in this in vitro method, two full-length parental bivalent antibodies are provided, each comprising a mutation in the antibody CH3 region that favors heterodimer formation resulting in bispecific antibodies containing a half-arm from each parental antibody. Mutations that may be used to favor heterodimer formation are F405L in one parental antibody and R409K in the other prenatal antibody for IgG1 antibodies, or F405L and K409R one parental antibody while retaining wild-type CH3 for IgG4 antibodies.

The monospecific antibodies were expressed in HEK cell lines under CMV promoters.

The parental antibodies were purified using a protein A column with an elution buffer of 100 mM NaAc pH3.5 and a neutralization puffer of 2M Tris pH 7.5 and 150 mM NaCl. The mabs were desalted using PD10 (Sephadex G25M) column and dialyzed into D-PBS, pH 7.2 buffer.

Post purification the parental antibodies were mixed under reducing conditions in 75 mM cysteamine-HC1 and incubated at 31° C. for 4 h. The recombination reactions were based on molar ratios, where a set amount of target parent (eg, 10 mg, or ˜71.8 nanomoles) was combined with CD3 antibody (eg, ˜67.8 nanomoles), where the target parent antibody was added in a 6% excess of the CD3 antibody. The recombinations were subsequently dialyzed against PBS to remove the reductant. The bispecific antibody reactions were performed with an excess of the target parent antibody (ratio) to minimize the amount of unreacted CD3 parental antibody remaining after recombination. Following the partial reduction of the parental mAbs, the reductant was removed via overnight dialysis into PBS.

Results: FIG. 10 shows the inhibition curves for the bivalent and monovalent anti CD3 constructs, CD3B376 and CD3B450, competing for binding against the AlexaFluor488 SP-34 anti CD3 tracer antibody. Increasing concentrations of the test anti CD3 antibodies decreased the binding of the AlexaFluor488 tracer antibody, thus decreasing the mean fluorescence intensity (MFI). IC50 values were generated, and using the aforementioned formula, K_daffinities were calculated and summarized in Table 13.

The CD3B376 binding site was tighter than the CD3B450 in both bivalent and monovalent form.

TABLE 13

IC50 and K_daffinity values of the anti CD3 bivalent

and monovalent CD3B376 and CD3B450 constructs

Construct
anti CD3
IC50 (nM)
K_d(nM)

Bivalent
CD3B376
29
7.3

CD3B450
60
15

Monovalent
CD3B376
409
103

Construct
CD3B450
1011
254

1-10 Conformational Stability of Anti-CD3 Monoclonal Antibodies

Conformational stability of the anti-CD3 antibodies CD3B376, CD3B389 (IgG1 sigma version of CD3B376; heavy chain is SEQ ID NO: 729; light chain is SEQ ID NO: 676), CD3B450, and CD3B467 (IgG1 sigma version of CD3B450; heavy chain is SEQ ID NO: 728; light chain is SEQ ID NO: 677) was determined by differential scanning calorimetry (DSC). The mid-point of thermal transition, Tm, was determined from the thermal denaturation profiles of each of the Ab candidates. FIG. 12A-FIG. 12E show the thermal denaturation profiles of the anti-CD3 antibodies in PBS. Table 14 includes a summary of the Tm and the enthalpy values (ΔH) for the thermal unfolding of the anti-CD3 antibodies as determined by DSC.

The DSC results indicate that all anti-CD3 antibodies CD3B376, CD3B389, CD3B450, and CD3B467 have folded domains. Based on the onset of unfolding, the relative stability of each antibody was as follows: CD3B389<CD3B467<CD3B376<CD3B450. The anti-CD3 molecules tested by DSC displayed some differences in their thermal stability. The CD3B376 (IgG4 PAA) molecule displayed three partially unresolved transitions at 59.7, 62.4 and 69.2° C. with a total enthalpy of unfolding of 417.6 kcal/mol, while the CD3B450 (IgG4 PAA) molecule displayed two unresolved transitions at 62.5 and 66.3° C. with a total enthalpy of unfolding of 545.1 kcal/mol. The CD3B389 (IgG1sigma) molecule displayed four transitions at 54.6, 58.2, 73.1 and 77.1° C. with a total enthalpy of unfolding of 401.7 kcal/mol, while the CD3B467 (IgG1sigma) molecule displayed four transitions at 56.3, 59.6, 66.5 and 75.6° C. with a total enthalpy of unfolding of 406.2 kcal/mol.

TABLE 14

Summary of the thermal transition data for the anti-CD3 antibodies in PBS. Values represent averages of

duplicate runs. Sequence information is provided for HC and LC peptides (SEQ ID NOs in parentheses).

CD3B376
CD3B450
CD3B389
CD3B467

HC: SEQ ID NO: 640
HC: SEQ ID NO: 675
HC: SEQ ID NO: 729
HC: SEQ ID NO: 728

LC: SEQ ID NO: 676
LC: SEQ ID NO: 677
LC: SEQ ID NO: 676
LC: SEQ ID NO: 677

Average
Error
Average
Error
Average
Error
Average
Error

Tm1 (° C.)
59.7
0.1
62.5
0.9
54.6
0.1
56.3
0.1

ΔH1
151900.0
4200.0
158175.0
64025.0
166150.0
2450.0
98160.0
17840.0

(cal/mol)

Tm2 (° C.)
62.4
0.1
66.3
0.2
58.2
0.1
59.6
0.0

ΔH2
202200.0
4300.0
386900.0
71000.0
62050.0
1290.0
72900.0
370.0

(cal/mol)

Tm3 (° C.)
69.2
0.0
NA
NA
73.5
0.1
66.5
0.0

ΔH3
63450.0
2080.0
NA
NA
101900.0
1600.0
92785.0
6785.0

(cal/mol)

Tm4 (° C.)
NA
NA
NA
NA
77.1
0.0
75.6
0.1

ΔH3
NA
NA
NA
NA
71560.0
2500.0
142400.0
4800.0

(cal/mol)

Total ΔH
601000.0
NA
545075.0
NA
401660.0
NA
406245.0
NA

The two IgG1 antibodies display lower stability compared to the corresponding IgG4 PAA antibodies (comparing the molecules with the same variable domains) with Tm of the first transition being 5-6° C. lower (FIG. 13A and FIG. 13B and Table 14).

1-11 Crystal Structure of CD3B334 Fab in Complex with CD3e N-Terminal Peptide

Anti-CD3 mAb CD3B334 (CD3H231/CD3L137) was modified to increase the “humanness” index by replacing a number of framework residues in the antibody by the human germline residues. This procedure yielded antibody CD3B334 with the following mutations: D43G/L49M/L50I/S62N/Q85E/H89Y in VL when compared to the parental VL CD3L124 and R10G/R13K/V73I/R79K/T83S/L96V in VH when compared to the parental VH CD3H219. The His-tagged Fab fragment of CD3B334 was expressed in HEK 293Expi cells and purified using affinity and size-exclusion chromatography. The N-terminal 9-mer peptide of human CD3e was synthesized at New England Peptide (Lot V1108-19/21) and mixed with Fab at molar ratio 10:1 (excess of peptide). The complex was crystallized by the vapor-diffusion method from solution containing 4 M Na formate in 0.1 M Tris, pH 8.5. The crystals belong to the orthorhombic space group P212121 with unit cell dimensions of 66.5×69.4×100.4 Å and one complex molecule in the asymmetric unit. The structure of the complex was determined at 1.8 Å resolution by the molecular replacement method using the crystal structure of the Fab as a search model.

CD3B334 bound residues 1-6 of CD3e. The N-terminal Gln of the peptide was in the pyroglutamate form and in a hydrophobic environment, between F107 of HCDR3 and L99 of LCDR3. Two arginine residues, R52 and R56, from HCDR2 were engaged in electrostatic interactions with the acidic residues of CD3. In total, 16 residues formed the CD3B334 paratope. Residues from all CDRs except LCDR2 were in direct contact (within 4 Å distance) with the CD3 peptide (see FIG. 18).

2 PSMA Antibodies
2-1 Generation of PSMA Cell Lines

Expression vectors presenting full-length chimpanzee PSMA (H2Q3K5_PANTR, SEQ ID NO: 49) or full length Cynomolgous monkey PSMA (EHH56646.1, SEQ ID NO: 50) were generated for use as screening tools to assess the anti-PSMA leads using an in-house expression vector with the CMV promoter using standard molecular biology techniques. Vectors were transiently transfected into HEK293F cells in suspension using standard methods. Transfected 293F suspension cells were plated in growth medium plus serum to become adherent and selected for stable plasmid integration. Single cell populations were selected by serial dilution and the PSMA surface receptor expression was quantified by FACS using the (PSMAL antibody (Center) affinity Purified Rabbit Polycolonal Antibody (Catalog #OAAB02483, Aviva Systems Biology) as the primary antibody with a R-PE anti-rabbit secondary antibody (Catalog #111-116-144, Jackson ImmunoResearch Laboratories, Inc.) and a rabbit polyclonal IgG (Catalog #SC-532, Santa Cruz Biotechnology) as the isotype control).

Human PSMA expressing cell lines were generated using lentivirus (Genecopoeia, cat #EX-G0050-Lv105-10) containing full length human PSMA (FOLH1_HUMAN, SEQ ID NO:51) and puromycin for selection of PSMA positive cells. HEK293F cells (ATCC), negative for PSMA, were transduced with Lentiviral particles to overexpress human PSMA. Following transduction, cells positively expressing PSMA and the resistance marker were selected by treating pooled cells, grown in DMEM+10% HI FBS (Life Technologies) and supplemented with varying concentrations of Puromycin (Life Technologies).

In addition to the HEK generated cell lines, several commercial cell lines were used for phage panning and binding and cellular toxicity assays. LNCaP clone FGC cells (ATCC cat #CRL-1740) are a commercially available human prostate cancer cell lines. C4-2B cells were originally developed at MD Anderson and are derived from LNCaP FGC grown in vivo and metastasize to bone marrow (Thalmann, et al 1994, Cancer Research 54, 2577-81).

2-2 Generation of Soluble PSMA ECD Proteins

Recombinant chimpanzee PSMA Extra Cellular Domain (ECD) protein (Chimp PSMA ECD, SEQ ID NO:52) was generated for panning and to assess the anti-PSMA leads using an in-house expression vector with the CMV promoter using standard molecular biology techniques. The chimp PSMA ECD gene fragment (amino acid 44-750 of SEQ ID NO:49) with N-terminal signal sequence (SEQ ID NO:594), N-terminal Avitag (SEQ ID NO:595) and 6-His tags (SEQ ID NO:596) was cloned using an in-house expression vector with the CMV promoter using standard molecular biology techniques and transiently expressed in 293Expi cells (Invitrogen). cDNA was prepared using gene synthesis techniques (U.S. Pat. Nos. 6,670,127; 6,521,427). Supernatants were harvested and clarified by centrifugation. The proteins were purified using a two-step purification process: 1) IMAC purification with a HisTrap HP column (GE Healthcare) and 2) size exclusion purification (Superdex 200, Ge Healthcare) where the elution buffer is Dulbecco's phosphate-buffered saline, calcium, magnesium (Thermofisher, #14040) containing 0.5 mM ZnCl₂to stabilize PSMA dimerization. Fractions containing the protein of interest were pooled and protein concentration was determined by A280. This material was used for binding and affinity measurements and is referred to as PSMG8.

Chimp PSMA ECD was also biotinylated for panning. The BirA plasmid that was co-transfected into mammalian cells to biotinylate proteins containing the Avi tag was created in-house. The BirA coding region (SEQ ID NO:597) was fused to the signal peptide from mouse IgG heavy chain (SEQ ID NO:598), and an ER retention signal (KDEL (SEQ ID NO: 716)) was added to the C-terminus to generate the BirA plasmid (SEQ ID NO:599). The constructed gene was cloned into an expression vector under the control of the CMV promoter. To produce biotinylated PSMA antigen, the PSMA plasmid DNA was added in a 4-fold excess (w/w) to the BirA plasmid into the transfection mix.

Biotinylation of the Chimp PSMA ECD protein was performed via the Avi tag by cotransfection of a BirA expression construct and the resulting secreted protein was purified using a two-step purification process: 1) IMAC purification with a HisTrap HP column (GE Healthcare) and 2) size exclusion purification (Superdex 200, Ge Healthcare) where the elution buffer is Dulbecco's phosphate-buffered saline, calcium, magnesium (Thermofisher, #14040) containing 0.5 mM ZnCl2 to stabilize PSMA dimerization. The protein was tested for endotoxin prior to use in phage panning studies.

Recombinant cynomolgous monkey PSMA extracellular domain (ECD) protein (cyno PSMA ECD, SEQ ID NO:53), corresponding to amino acids 44-750 of SEQ ID NO:50 with N-terminal signal (SEQ ID NO:594), N-terminal Avi- (SEQ ID NO:595) and 6His- (SEQ ID NO:596) tags was cloned and expressed as described previously for the chimp PSMA ECD. Biotinylation of the cyno PSMA ECD protein was performed via the Avi tag by cotransfection of a BirA expression construct and the resulting secreted protein was purified by a two-step purification using IMAC HisTrap HP column (GE Healthcare) and MonoAvidin columns. The protein was tested for endotoxin prior to use in phage panning studies. This material was also used for binding and affinity measurements and is referred to as PSMG1.

A second recombinant cyno PSMA ECD protein (Cyno PSMA Fc, SEQ ID NO:54) with an IgG1 Fc (SEQ ID NO:593) was cloned and expressed using an in-house expression vector with the CMV promoter using standard molecular biology techniques. CynoPSMA Fc protein was transiently expressed in 293HEK-expi cells. Transient transfection of PSMG3 in HEK293 Expi cells were harvested 5 days after transfection, clarified by centrifugation (30 min, 6000 rpm) and filtered (0.2μ PES membrane, Corning). The relative amount of IgG was determined with the Octet instrument (ForteBio) using a purified known IgG (same isotype) spiked into spent medium to generate the standard curve.

Clarified Cyno PSMA Fc supernatant was loaded onto an equilibrated (dPBS, pH 7.2) HiTrap MabSelect Sure Protein A column (GE Healthcare) at a relative concentration of ˜30 mg protein per ml of resin. After loading, the column was washed with dPBS, pH7.2 and protein eluted with 10 column volumes of 0.1 M Na-Acetate, pH 3.5. Peak fractions were pooled, neutralized with 2M Tris, pH 7, and filtered (0.20. The neutralized protein sample was dialyzed against 3 changes of dPBS containing Ca2+, Mg2+, and 0.5 mM ZnCl2, pH 7.2 overnight at 4° C. The next day, sample was removed from dialysis, filtered (0.2μ) and the protein concentration determined by absorbance at 280 nm on a BioTek SynergyHT™ spectrophotometer. The quality of the purified proteins was assessed by SDS-PAGE and analytical size exclusion HPLC (Dionex HPLC system). Endotoxin levels were measured using a LAL assay (Pyrotell-T, Associates of Cape Cod). Purified proteins were stored at 4° C.

Recombinant human PSMA extracellular domain (ECD) protein (human PSMA ECD, SEQ ID NO:55), corresponding to amino acids 44-750 of SEQ ID NO:51 with N-terminal Avi- and 6His tags (SEQ ID NO: 596) was cloned, expressed and purified as described previously for the chimp and cyno PSMA ECD proteins.

2-3 Identification of Anti-Chimp and Anti-Human PSMA Fabs
Panning with Recombinant Protein

A first solution panning of the de novo Human Fab-pIX libraries [Shi, L., et al J Mol Biol, 2010. 397(2): p. 385-396. WO 2009/085462], consisting of VH1-69, 3-23 and 5-51 heavy chain libraries paired with four human VL germline genes (A27, B3, L6, O12) libraries, was performed using an alternating panning approach with one round of phage capture on Strepavidin beads (Invitrogen Cat #112.05D, Lot #62992920) coated with biotinylated Chimp PSMA ECD according to the manufacturer's protocol, followed by phage capture on ProtG beads (Invitrogen, Cat #10003D) coated with Cyno-PSMA-Fc according to the manufacturer's protocol followed by phage capture on Sera-mag Double Speed magnetic Neutravidin beads (Thermo, Cat #7815-2104-011150) coated with biotinylated Chimp PSMA ECD according to the manufacturer's protocol. This panning yielded two hits: PSMM18 and PSMM25.

Whole Cell Panning for Anti-PSMA Fabs

Additional panning experiments were performed on whole cells using the Round #1 output from the chimpanzee ECD panning experiments described above or fresh de novo phage libraries, as input. Briefly, phage was produced by helper phage infection and concentrated by PEG/NaCl precipitation according to standard protocols known in the art. The phage libraries were pre-cleared on untransfected parental HEK293F cells overnight at 4° C. with gentle rocking. Following PEG/NaCl precipitation, the pre-cleared libraries were incubated with chimp PSMA expressing HEK293 cells or LNCAP cells with gentle rocking for 2 hr at 4° C. The removal of unbound phage and the recovery of phage-bound cells was performed by Ficoll gradient, and following several wash steps with, cells carrying bound phage were incubated with 1 mL of TG-1 E. coli culture at 37° C. for 30 minutes without agitation. The resulting mixture was plated on LB-Carbenicillin-1% Glucose plates and grown over night at 37° C. The process was then repeated for subsequent panning rounds.

Conversion of Phage Fab-pIX to Fab-his for Generating E. coli Supernatants

The resulting phage Fab-pIX hits were converted to Fab-His using a standard procedure. Plasmid DNA was isolated from phage panned E. coli (Plasmid Plus Maxi Kit, Qiagen cat #12963) and subjected to NheI/SpeI restriction digest. The resulting 5400 and 100 bp fragments were separated on a 0.8% agarose gel and the 5400 bp fragment was gel purified (MinElute PCR purification kit, Qiagen cat #28006). The purified 5400 bp band was self-ligated using T4 ligase and the resulting product (encoding the Fab-his fusion) was transformed back into the TG-1 E. coli strain and clonally isolated. Fab-His supernatants were generated from clones by overnight induction of cultures with 1 mM IPTG. Following centrifugation of the overnight culture, clarified supernatants were ready for use in downstream assays. To determine the relative expression levels of different Fab-his supernatants, an anti-kappa (Southern Biotech cat #2061-05) ELISA on serially diluted supernatants was performed. All of the clones tested exhibited similar Fab-his expression (data not shown).

Cell Binding of Fab-his Fusions from E. coli

A cell-based binding assay was designed to assess the binding capabilities of individual Fab-his fusions from E. coli supernatants to PSMA-expressing cells. Individual Fab clones were isolated from the round 3 output of all panning experiments following pIX excision. Fab clones were tested for binding to chimp and cyno PSMA expressing HEK cells, as well as to human PSMA on LNCaP cells. Briefly, PSMA expressing cells were aliquoted into a V-bottom plate (CoStar 3357) at a density of 200,000 per well and incubated with (100 μl) supernatants expressing Fab fragments for 1 hour on ice. Cells were washed twice with PBS containing 2% FBS, and stained with a mouse anti-human kappa-RPE antibody (Life Technologies cat #MH10514) for 1 hour on ice. Cells were washed twice with PBS containing 2% FBS and resuspended in 1004 of the same wash buffer. Plates were read on a BD FACS Array flow cytometer. FACS data was analyzed in FlowJo software by live gating the healthy population of cells using forward scatter and side scatter, and then analyzing the cells within this gate for PE staining. Mean fluorescence intensity (MFI) was calculated and exported into Microsoft Excel. Fab clones that exhibited binding 3 times background for all three species of PSMA (cyno, chimp and human), and exhibited no binding to the HEK293 cell line, were labeled as “preliminary positive”. Fabs were sequenced and moved forward for cloning into mammalian expression vector for rescreening. True positives were selected from the binding of mammalian cell expressed Fab supernatants to PSMA-expressing cell lines.

Preparation of Mammalian Fabs

For conversion of E. coli Fab to mammalian-expressed Fab, In-Fusion HD cloning (ClonTech cat #638918) was utilized according to the manufacturer's protocol. Briefly, nucleotide sequences of clones that have passed the primary screen and are to be moved into mammalian Fab format, are loaded into the “InFu Primer Finder v1.2.3” program (software developed in-house), which generates a list of isotype-specific PCR primers used to generate PCR fragments for In-Fusion cloning into the huKappa_muIgGSP and huG1 Fab expression vectors. These vectors are in-house vectors with CMV promotors based off of pcDNA3.1. Following the In-fusion process, E. coli clones were isolated, sequence verified and transfected into HEK293 cells using standard protocols. Mammalian PSMA Fabs for confirming binding to PSMA expressing cell lines were prepared by harvesting 20 ml of supernatants from transfection after 5 days.

Rescreening Hits from Whole Cell Panning in Mammalian Sup Format

Confirmation of mammalian expressed Fab supernatants was performed using the whole cell binding assay described previously. Binding of Fabs to human PSMA (LNCaP), chimpanzee, and cynomolgous monkey cells was tested, as well as counter screening for no binding to the parental HEK cell line. Table 19 shows the hit profile of mammalian Fab supernatant binding to PSMA-expressing cells. Many of the hits from E. coli supernatants did not confirm with mammalian expressed proteins. PSMM48 showed high binding to cyno PSMA-expressing cells and some binding to chimp-PSMA expressing cells, but no binding to LNCaP cells expressing human PSMA. PSMM56 showed a similar profile, but with some binding to LNCaP cells. PSMM69-80 bound to LNCaP cells, but not to chimp- or cyno-PSMA expressing cells. Mammalian Fab sups PSMM52, M56 and M57, bound all three cell lines. PSMM50, M51, and M54, show more chimp or cyno binding. M58 showed slight chimp and cyno binding.

TABLE 19

Hit profile of Mammalian Fab protein binding to PSMA-expressing

cells measured by Geo-MFI (Mean Fluoresent Instensity)

Fab ID (Fab DNA ID)
cyno
chimp
LNCaP
Parent HEK

PSMB10 (PSMM10)
244
81.6
—
248

PSMB11 (PSMM11)
19
6.6
—
8.14

PSMB12 (PSMM12)
31.6
8.05
—
12.6

PSMB13 (PSMM13)
57.8
18.2
—
50.5

PSMB14 (PSMM14)
32.6
13.1
—
22.2

PSMB15 (PSMM15)
40.4
18.5
—
38

PSMB16 (PSMM16)
175
220
—
6.39

PSMB17 (PSMM17)
34.9
22.4
—
40.1

PSMB18 (PSMM18)
696
439
—
8.71

PSMB19 (PSMM19)
53.7
—
5.15
4.47

PSMB20 (PSMM20)
5.75
—
5.85
41.3

PSMB21 (PSMM21)
94.4
—
20.7
372

PSMB22 (PSMM22)
9.07
—
7.92
54.9

PSMB23 (PSMM23)
16.4
—
6.66
164

PSMB24 (PSMM24)
14.6
9.6
4.09
3.96

PSMB25 (PSMM25)
15.2
11.3
16.9
4.09

PSMB26 (PSMM26)
9.48
—
7.26
114

PSMB27 (PSMM27)
20
—
7.56
136

PSMB28 (PSMM28)
29.7
—
8.88
302

PSMB29 (PSMM29)
6.87
—
5.7
72.8

PSMB30 (PSMM30)
5.16
—
4.58
45

PSMB31 (PSMM31)
5.99
—
—
25.5

PSMB32 (PSMM32)
4.81
—
—
27.1

PSMB33 (PSMM33)
5.14
—
—
40.1

PSMB34 (PSMM34)
17.9
—
—
107

PSMB35 (PSMM35)
58.5
—
—
231

PSMB36 (PSMM36)
5.05
—
—
6.96

PSMB37 (PSMM37)
23.4
—
—
178

PSMB38 (PSMM38)
4.05
—
—
7.7

PSMB39 (PSMM39)
10.2
—
—
166

PSMB40 (PSMM40)
66.9
—
—
348

PSMB41 (PSMM41)
5.39
—
—
12

PSMB42 (PSMM42)
7.35
—
—
25.8

PSMB43 (PSMM43)
8.73
—
—
7.18

PSMB44 (PSMM44)
12.6
—
—
48.9

PSMB45 (PSMM45)
22.4
—
—
43.1

PSMB46 (PSMM46)
3.88
—
—
5.29

PSMB47 (PSMM48)
101
25.5
3.46
2.85

PSMB48 (PSMM49)
2.72
3.18
2.68
2.72

PSMB49 (PSMM50)
51.6
22
3.22
3.48

PSMB51 (PSMM52)
285
231
41.5
2.68

PSMB52 (PSMM53)
39.2
6.89
2.67
2.56

PSMB53 (PSMM54)
27.6
17.8
4
2.6

PSMB54 (PSMM55)
2.7
2.75
2.65
2.79

PSMB55 (PSMM56)
226
180
17.2
2.58

PSMB56 (PSMM57)
95.6
34.7
24.5
2.52

PSMB57 (PSMM58)
19.8
11
3.26
2.68

PSMB58 (PSMM59)
121
192
25.3
2.67

PSMB59 (PSMM60)
4.96
9.69
6.04
3

PSMB60 (PSMM61)
2.28
3.07
87.3
4.64

PSMB61 (PSMM62)
2.1
3.16
135
2.98

PSMB62 (PSMM63)
7.17
4.43
54.9
9.09

PSMB63 (PSMM64)
2.07
2.95
27
2.82

PSMB64 (PSMM65)
2.39
3.26
70.5
3.05

PSMB65 (PSMM66)
2.3
3.13
32.4
4.25

PSMB66 (PSMM67)
2.14
3
24.6
2.83

PSMB67 (PSMM68)
2.23
2.95
21
2.95

PSMB68 (PSMM69)
5.44
—
134
35.3

PSMB69 (PSMM70)
2.29
3.38
25.5
3.35

PSMB70 (PSMM71)
2.22
3.49
15.5
3.26

PSMB71 (PSMM72)
2.54
4.4
18.5
3.07

PSMB72 (PSMM73)
2.13
3.53
227
3.02

PSMB73 (PSMM74)
2.97
4.13
125
11.1

PSMB74 (PSMM75)
120
—
178
132

PSMB75 (PSMM76)
2.99
3.04
173
7.89

PSMB76 (PSMM77)
3.75
3.99
138
3.95

PSMB77 (PSMM78)
4.68
3.96
144
4.71

PSMB78 (PSMM79)
25.2
—
378
24.4

PSMB79 (PSMM80)
38.4
—
512
157

PSMB80 (PSMM81)
19.6
18.6
20.9
6.61

PSMB81 (PSMM82)
2.63
2.06
4.07
2.69

PSMB82 (PSMM83)
2.79
2.23
4.11
2.76

PSMB83 (PSMM84)
2.59
2.28
4.09
2.74

PSMB84 (PSMM85)
750
729
192
3.15

PSMB85 (PSMM86)
2.84
2.59
2.33
3.24

PSMB86 (PSMM87)
224
176
31.7
2.82

PSMB87 (PSMM88)
2.63
2.27
4.23
2.91

PSMB88 (PSMM89)
37.7
29.7
30.3
7.6

PSMB89 (PSMM90)
27.1
27.3
53.2
39.5

PSMB90 (PSMM91)
26.7
24.7
47.1
36.4

PSMB91 (PSMM92)
8.97
6.16
13
6.63

PSMB92 (PSMM93)
20
16.5
57.1
50

PSMB93 (PSMM94)
5.13
9.62
2.5
3.66

PSMB94 (PSMM95)
5.12
2.67
2.22
3.57

PSMB95 (PSMM96)
8.9
8.82
13.4
11.4

PSMB96 (PSMM97)
2.4
3.25
2.53
4.03

PSMB97 (PSMM98)
2.57
4.73
2.52
3.7

PSMB99 (PSMM100)
9.95
2.4
2.39
4.03

PSMB100 (PSMM101)
4.03
2.52
2.33
3.37

PSMB100 (PSMM101)
3.5
2.86
2.48
4.57

PSMB101 (PSMM102)
5.49
3.18
2.23
3.33

PSMB102 (PSMM103)
2.4
2.42
2.16
3.2

PSMB103 (PSMM104)
3.52
3.26
2.58
4.44

PSMB104 (PSMM105)
2.15
2.5
2.34
3.95

PSMB105 (PSMM106)
2.03
2.39
2.18
3.39

PSMB106 (PSMM107)
2
2.4
2.27
3.59

PSMB107 (PSMM108)
2
2.47
2.33
3.49

PSMB108 (PSMM109)
2
2.58
2.28
3.46

PSMB109 (PSMM110)
321
326
34.9
6.11

PSMB110 (PSMM111)
2.3
2.31
2.31
3.4

PSMB111 (PSMM112)
2.32
2.31
—
3.21

PSMB112 (PSMM113)
6.28
5.7
2.71
3.28

PSMB113 (PSMM114)
2.82
2.95
2.32
3.29

PSMB114 (PSMM115)
2.78
2.47
4.3
3.14

PSMB115 (PSMM116)
2.66
2.59
2.2
3.14

PSMB46 (PSMM117)
4.54
3.18
2.21
4.79

PSMB67 (PSMM118)
3.95
4.3
3
6.13

PSMB74 (PSMM119)
7.94
13
3.16
12.5

PSMB78 (PSMM120)
5.08
4.79
22.3
6.82

PSMB81 (PSMM121)
3.66
3.83
3.05
5.11

PSMB82 (PSMM122)
15.1
28.4
10.8
24.3

PSMB83 (PSMM123)
37.5
42.1
3.04
4.88

PSMB85 (PSMM124)
34.6
52.9
20.7
46.8

PSMB87 (PSMM125)
4.23
3.74
2.26
4.73

PSMB89 (PSMM126)
51.8
53.1
11.7
6.27

PSMB90 (PSMM127)
42.8
30.2
7.74
5.99

PSMB91 (PSMM128)
3.9
27.6
2.37
4.32

PSMB92 (PSMM129)
45.7
37.3
12.1
7.4

PSMB93 (PSMM130)
5.13
7.85
4.11
7.82

PSMB94 (PSMM131)
3.67
3.23
2.32
4.72

PSMB95 (PSMM132)
4.05
3.64
2.56
5.57

PSMB96 (PSMM133)
3.91
4.54
2.37
4.65

PSMB97 (PSMM134)
3.22
3.16
4.08
4.22

PSMB98 (PSMM135)
15.6
12.7
2.22
4.21

PSMB99 (PSMM136)
4.08
3.26
2.22
5.04

PSMB100 (PSMM137)
5.24
3.82
2.16
4.83

PSMB101 (PSMM138)
3.84
3.14
2.23
4.52

PSMB102 (PSMM139)
4.51
3.82
2.23
4.59

PSMB103 (PSMM140)
6.81
4.27
2.21
5.41

PSMB104 (PSMM141)
7.52
4.35
2.26
4.39

PSMB105 (PSMM142)
5.03
11.2
4.87
7.28

PSMB106 (PSMM143)
3.87
3.8
2.73
4.9

PSMB107 (PSMM144)
3.3
3.35
2.3
4.64

PSMB108 (PSMM145)
6.78
3.83
2.33
4.98

PSMB110 (PSMM146)
4.03
3.23
2.28
5.3

PSMB111 (PSMM147)
3.71
3.26
2.36
5.11

PSMB112 (PSMM148)
4.54
3.26
2.26
4.86

PSMB113 (PSMM149)
84.3
104
51.7
94.2

PSMB114 (PSMM150)
3.31
3.26
2.21
5.14

PSMB115 (PSMM151)
3.55
3.43
2.3
4.21

Dose Response Curves of Mammalian Expressed Fabs

Once mammalian expressed Fab clones were confirmed for positive binding as neat Fab supernatants to PSMA expressing cell lines, the supernatants were normalized for protein concentration by Octet or protein gel, and dose-response curves were completed to confirm PSMA binding using the protocol described previously. FIG. 22-FIG. 24 show titration curves for hits that demonstrated binding to all three PSMA-expressing cells. FIGS. 22A, 22B, 22C and 22D show the titration curves for anti-PSMA panning hits vs. LNCaP cells. FIGS. 23A, 23B, 23C, and 23D show the titration curves for anti-PSMA panning hits vs chimp-PSMA HEK cells. FIGS. 24A, 24B, 24C, and 24D show the titration curves for anti-PSMA panning hits vs Cyno-PSMA HEK cells. Binding profiles among hits were compared across cell lines expressing different species of PSMA. PSMM52 supernatant was used as a positive control across experiments. Several hits were deprioritized because of N-linked glycosylation sites in CDRs, binding to the PSMA negative parental HEK cell line, or lack of binding to PSMA positive cell lines. Eleven Fab hits remained and 10 hits were cloned into human IgG4-PAA heavy chain constructs and used to generate PSMA×CD3 bispecific antibodies. These hits showed cross-species binding within 3-fold of each other and were moved into a bispecific antibody format to be tested for T cell redirection killing of PSMA positive targets. The panning antigens for each hit is shown in Table 20.

TABLE 20

Antigen for each of the panning hits

Round 2-3

Round 1 antigen
antigen
Hits
Hit identification

Chimp PSMA ECD
Cyno PSMA
2
PSMM18, PSMM25

ECD

Chimp PSMA ECD
Chimp PSMA
9
PSMM50, PSMM52,

HEK

PSMM57, PSMM59,

PSMM110, PSMM56,

PSMM85, PSMM84

LNCaP
Chimp PSMA
2
PSMM87, PSMM81

HEK

Preparation of Anti-PSMA mAbs

A total of 12 clones that demonstrated binding to all three PSMA-expressing cells were ultimately converted to mAb IgG4 having Fc substitutions S228P, F234A, and L235A (PAA) isotype by restriction cloning. Briefly, constructs corresponding to Fab clones that have passed initial screens were digested with HindIII and ApaI. Gel purified fragments were ligated into an expression vector with CMV promotervDR000215, a CMV driven expression vector containing the human IgG4-PAA Fc for full mAb expression. This allowed for rapid generation of bispecific antibodies. The expression vector previously described was used to express the Heavy and Light Chains for each PSMA mab, where both vectors were co-transfected transiently into 293Expi or CHO cell lines for expression of the mAb. CDR sequences of cross-species positive PSMA Fabs generated from phage panning are shown below in Table 21. VH and VL sequences of the selected Fabs are shown below in Table 22. Heavy and light chain sequences of mAbs generated from the Fabs are shown in Table 23.

TABLE 21

CDR sequences (defined according to Kabat) of FAbs from phage panning

(corresponding SEQ ID NOs are listed in parentheses)

CDRs (SEQ ID NO:)

FAB ID

CDR1
CDR2
CDR3

PSMB109
HC
NAWIS
WINPESGRANYAQKFQG
ELYYLVYSTYYYAFDY

(62)
(63)
(64)

LC
RASQSIDRWLN
AASSLQS
QQSPRYPLT

(65)
(60)
(66)

PSMB86
HC
SYDIS
GIIPIEGTANYAQKFQG
DYPAGYGFDY

(67)
(68)
(69)

LC
RASQSVSSSYLA
GASSRAT
QQYGSSPLT

(70)
(71)
(72)

PSMB84
HC
SDWMS
AISGNGGSTEYADSVKG
DPYYYYDGDSYYGMDV

(73)
(74)
(72)

LC
RASQSISSYLN
AASSLQS
QQSYSTP

(76)
(60)
(61)

PSMB83
HC
SDAMH
EISGSGGYTNYADSVKG
DSYDSSLYVGDYFDY

(78)
(79)
(80)

LC
RASQSVSSYLA
DASNRAT
QQRSNWPLT

(81)
(82)
(83)

PSMB56
HC
SYAIS
WISPYNGNANYAQKFQG
DSDRSYNLDY

(84)
(85)
(86)

LC
RASQSISGWLN
AASSLQS
QQSYSTPLT

(87)
(60)
(88)

PSMB55
HC
SYWIG
IIYPGDSDTRYSPSFQG
GLPIWYLDY

(89)
(90)
(91)

LC
RASQSVASDLA
FASNRAT
QQSITWPFT

(92)
(93)
(94)

PSMB51
HC
SYAIS
WIIPYNGNANYAQKFQG
VNSAALVWERLDY

(95)
(96)
(97)

LC
RASQSIDRWLN
AASSLQS
QQSPRYPLT

(65)
(60)
(66)

PSMB49
HC
SYAIS
GIIPIFGTANYAQKFQG
ASRVWHASYGYLDY

(84)
(98)
(99)

LC
RASQSVSKWLA
DASNRAT
QQRFTAPWT

(100)
(82)
(101)

PSMB25
HC
SYWIG
IIYPGDSDTRYSPSFQG
GWAYDRGLDY

(89)
(90)
(102)

LC
KSSQSVLYSSNNKNYLA
WASTRES
QQYYSTPLT

(103)
(104)
(105)

PSMB18
HC
SYWIG
IIYPGDSDTRYSPSFQG
AYHYSKGLDY

(89)
(90)
(106)

LC
KSSQSVLYSSNNKNYLA
WASTRES
QQYYSTPLT

(103)
(104)
(105)

PSMB80
HC
DYAIS
RIDPIEGTANYAQKFQG
DRYYYDGVYWYSDYFDY

(107)
(108)
(109)

LC
RASQSISSYLN
AASSLQS
QQSYSTPLT

(76)
(60)
(88)

PSMB58
HC
SYWIS
IIYPGDSYTRYSPSFQG
DYEWELFDSRLDY

(56)
(57)
(58)

LC
RASQSISSYLN
AASSLQS
QQSYSTP

(59)
(60)
(61)

TABLE 22

VH and VL sequences of PSMA Fabs

VL Amino

FAB ID
VH Amino acid sequence
SEQ ID NO
Acid Sequence
SEQ ID NO

PSMB109
QVQLVQSGAEVKKPGSSVKVSCKASGGTF
110
DIQMTQSPSSLSASVGDRV
111

SSYAISWVRQAPGQGLEWMGWISPYNGNA

TITCRASQSIDRWLNWYQQ

NYAQKFQGRVTITADESTSTAYMELSSLR

KPGKAPKLLIYAASSLQSG

SEDTAVYYCARVNSAALVWERLDYWGQGT

VPSRFSGSGSGTDFTLTIS

LVTVSS

SLQPEDFATYYCQQSPRYP

LTFGQGTKVEIK

PSMB86
QVQLVQSGAEVKKPGSSVKVSCKASGGTF
112
EIVLTQSPGTLSLSPGERA
113

KSYDISWVRQAPGQGLEWMGGIIPIEGTA

TLSCRASQSVSSSYLAWYQ

NYAQKFQGRVTITADESTSTAYMELSSLR

QKPGQAPRLLIYGASSRAT

SEDTAVYYCARDYPAGYGFDYWGQGTLVT

GIPDRFSGSGSGTDFTLTI

VSS

SRLEPEDFAVYYCQQYGSS

PLTFGQGTKVEIK

PSMB84
EVQLLESGGGLVQPGGSLRLSCAASGFTF
114
DIQMTQSPSSLSASVGDRV
115

DSDWMSWVRQAPGKGLEWVSAISGNGGST

TITCRASQSISSYLNWYQQ

EYADSVKGRFTISRDNSKNTLYLQMNSLR

KPGKAPKLLIYAASSLQSG

AEDTAVYYCARDPYYYYDGDSYYGMDVWG

VPSRFSGSGSGTDFTLTIS

QGTLVTVSS

SLQPEDFATYYCQQSYSTP

LTFGQGTKVEIK

PSMB83
EVQLLESGGGLVQPGGSLRLSCAASGFTF
116
EIVLTQSPATLSLSPGERA
117

KSDAMHWVRQAPGKGLEWVSEISGSGGYT

TLSCRASQSVSSYLAWYQQ

NYADSVKGRFTISRDNSKNTLYLQMNSLR

KPGQAPRLLIYDASNRATG

AEDTAVYYCARDSYDSSLYVGDYFDYWGQ

IPARFSGSGSGTDFTLTIS

GTLVTVSS

SLEPEDFAVYYCQQRSNWP

LTFGQGTKVEIK

PSMB80
QVQLVQSGAEVKKPGSSVKVSCKASGGTF
118
DIQMTQSPSSLSASVGDRV
119

DDYAISWVRQAPGQGLEWMGRIDPIEGTA

TITCRASQSISSYLNWYQQ

NYAQKFQGRVTITADESTSTAYMELSSLR

KPGKAPKLLIYAASSLQSG

SEDTAVYYCARDRYYYDGVYWYSDYFDYW

VPSRFSGSGSGTDFTLTIS

GQGTLVTVS

SLQPEDFATYYCQQSYSTP

LTFGQGTKVEIK

PSMB58
EVQLVQSGAEVKKPGESLKISCKGSGYSF
120
DIQMTQSPSSLSASVGDRV
115

TSYWISWVRQMPGKGLEWMGIIYPGDSYT

TITCRASQSISSYLNWYQQ

RYSPSFQGQVTISADKSISTAYLQWSSLK

KPGKAPKLLIYAASSLQSG

ASDTAMYYCARDYEWELFDSRLDYWGQGT

VPSRFSGSGSGTDFTLTIS

LVTVSS

SLQPEDFATYYCQQSYSTP

LTFGQGTKVEIK

PSMB56
QVQLVQSGAEVKKPGSSVKVSCKASGGTF
121
DIQMTQSPSSLSASVGDRV
122

SSYAISWVRQAPGQGLEWMGWISPYNGNA

TITCRASQSISGWLNWYQQ

NYAQKFQGRVTITADESTSTAYMELSSLR

KPGKAPKLLIYAASSLQSG

SEDTAVYYCARDSDRSYNLDYWGQGTLVT

VPSRFSGSGSGTDFTLTIS

VSS

SLQPEDFATYYCQQSYSTP

LTFGQGTKVEIK

PSMB55
EVQLVQSGAEVKKPGESLKISCKGSGYSF
123
EIVLTQSPATLSLSPGERA
124

TSYWIGWVRQMPGKGLEWMGIIYPGDSDT

TLSCRASQSVASDLAWYQQ

RYSPSFQGQVTISADKSISTAYLQWSSLK

KPGQAPRLLIYFASNRATG

ASDTAMYYCARGLPIWYLDYWGQGTLVTV

IPARFSGSGSGTDFTLTIS

SSA

SLEPEDFAVYYCQQSITWP

FTFGQGTKVEIK

PSMB51
QVQLVQSGAEVKKPGSSVKVSCKASGGTF
125
DIQMTQSPSSLSASVGDRV
111

SSYAISWVRQAPGQGLEWMGWIIPYNGNA

TITCRASQSIDRWLNWYQQ

NYAQKFQGRVTITADESTSTAYMELSSLR

KPGKAPKLLIYAASSLQSG

SEDTAVYYCARVNSAALVWERLDYWGQGT

VPSRFSGSGSGTDFTLTIS

LVTVSS

SLQPEDFATYYCQQSPRYP

LTFGQGTKVEIK

PSMB49
QVQLVQSGAEVKKPGSSVKVSCKASGGTF
126
EIVLTQSPATLSLSPGERA
127

SSYAISWVRQAPGQGLEWMGGIIPIFGTA

TLSCRASQSVSKWLAWYQQ

NYAQKFQGRVTITADESTSTAYMELSSLR

KPGQAPRLLIYDASNRATG

SEDTAVYYCARASRVWHASYGYLDYWGQG

IPARFSGSGSGTDFTLTIS

TLVTVSS

SLEPEDFAVYYCQQRFTAP

WTFGQGTKVEIK

PSMB25
EVQLVQSGAEVKKPGESLKISCKGSGYSF
128
DIVMTQSPDSLAVSLGERA
129

TSYWIGWVRQMPGKGLEWMGIIYPGDSDT

TINCKSSQSVLYSSNNKNY

RYSPSFQGQVTISADKSISTAYLQWSSLK

LAWYQQKPGQPPKLLIYWA

ASDTAMYYCARGWAYDRGLDYWGQGTLVT

STRESGVPDRFSGSGSGTD

VSS

FTLTISSLQAEDVAVYYCQ

QYYSTPLTFGQGTKVEIK

PSMB18
EVQLVQSGAEVKKPGESLKISCKGSGYSF
130
DIVMTQSPDSLAVSLGERA
131

TSYWIGWVRQMPGKGLEWMGIIYPGDSDT

TINCKSSQSVLYSSNNKNY

RYSPSFQGQVTISADKSISTAYLQWSSLK

LAWYQQKPGQPPKLLIYWA

ASDTAMYYCARAYHYSKGLDYWGQGTLVT

STRESGVPDRFSGSGSGTD

VSS

FTLTISSLQAEDVAVYYCQ

QYYSTPLTFGQGTKVEIK

TABLE 23

Heavy and Light chain sequences of PSMA monoclonal

antibodies with corresponding SEQ ID NOs

Heavy Chain

Light Chain

mAb ID
Amino acid sequence
SEQ ID NO
Amino Acid Sequence
SEQ ID NO

PSMB129
QVQLVQSGAEVKKPGSSVKVSCKAS
132
DIQMTQSPSSLSASVGDRVTIT
133

(FAB
GGTFSSYAISWVRQAPGQGLEWMGW

CRASQSIDRWLNWYQQKPGKAP

PSMB109)
ISPYNGNANYAQKFQGRVTITADES

KLLIYAASSLQSGVPSRFSGSG

TSTAYMELSSLRSEDTAVYYCARVN

SGTDFTLTISSLQPEDFATYYC

SAALVWERLDYWGQGTLVTVSSAST

QQSPRYPLTFGQGTKVEIKRTV

KGPSVFPLAPCSRSTSESTAALGCL

AAPSVFIFPPSDEQLKSGTASV

VKDYFPEPVTVSWNSGALTSGVHTF

VCLLNNFYPREAKVQWKVDNAL

PAVLQSSGLYSLSSVVTVPSSSLGT

QSGNSQESVTEQDSKDSTYSLS

KTYTCNVDHKPSNTKVDKRVESKYG

STLTLSKADYEKHKVYACEVTH

PPCPPCPAPEAAGGPSVFLFPPKPK

QGLSSPVTKSFNRGEC

DTLMISRTPEVTCVVVDVSQEDPEV

QFNWYVDGVEVHNAKTKPREEQFNS

TYRVVSVLTVLHQDWLNGKEYKCKV

SNKGLPSSIEKTISKAKGQPREPQV

YTLPPSQEEMTKNQVSLTCLVKGFY

PSDIAVEWESNGQPENNYKTTPPVL

DSDGSFFLYSRLTVDKSRWQEGNVF

SCSVMHEALHNHYTQKSLSLSLGK

PSMB130
QVQLVQSGAEVKKPGSSVKVSCKAS
134
EIVLTQSPGTLSLSPGERATLS
135

(FAB
GGTFKSYDISWVRQAPGQGLEWMGG

CRASQSVSSSYLAWYQQKPGQA

PSMB86)
IIPIEGTANYAQKFQGRVTITADES

PRLLIYGASSRATGIPDRFSGS

TSTAYMELSSLRSEDTAVYYCARDY

GSGTDFTLTISRLEPEDFAVYY

PAGYGFDYWGQGTLVTVSSASTKGP

CQQYGSSPLTFGQGTKVEIKRT

SVFPLAPCSRSTSESTAALGCLVKD

VAAPSVFIFPPSDEQLKSGTAS

YFPEPVTVSWNSGALTSGVHTFPAV

VVCLLNNFYPREAKVQWKVDNA

LQSSGLYSLSSVVTVPSSSLGTKTY

LQSGNSQESVTEQDSKDSTYSL

TCNVDHKPSNTKVDKRVESKYGPPC

SSTLTLSKADYEKHKVYACEVT

PPCPAPEAAGGPSVFLFPPKPKDTL

HQGLSSPVTKSFNRGEC

MISRTPEVTCVVVDVSQEDPEVQFN

WYVDGVEVHNAKTKPREEQFNSTYR

VVSVLTVLHQDWLNGKEYKCKVSNK

GLPSSIEKTISKAKGQPREPQVYTL

PPSQEEMTKNQVSLTCLVKGFYPSD

IAVEWESNGQPENNYKTTPPVLDSD

GSFFLYSRLTVDKSRWQEGNVFSCS

VMHEALHNHYTQKSLSLSLGK

PSMB128
EVQLLESGGGLVQPGGSLRLSCAAS
136
DIQMTQSPSSLSASVGDRVTIT
137

(FAB
GFTFDSDWMSWVRQAPGKGLEWVSA

CRASQSISSYLNWYQQKPGKAP

PSMB84)
ISGNGGSTEYADSVKGRFTISRDNS

KLLIYAASSLQSGVPSRFSGSG

KNTLYLQMNSLRAEDTAVYYCARDP

SGTDFTLTISSLQPEDFATYYC

YYYYDGDSYYGMDVWGQGTLVTVSS

QQSYSTPLTFGQGTKVEIKRTV

ASTKGPSVFPLAPCSRSTSESTAAL

AAPSVFIFPPSDEQLKSGTASV

GCLVKDYFPEPVTVSWNSGALTSGV

VCLLNNFYPREAKVQWKVDNAL

HTFPAVLQSSGLYSLSSVVTVPSSS

QSGNSQESVTEQDSKDSTYSLS

LGTKTYTCNVDHKPSNTKVDKRVES

STLTLSKADYEKHKVYACEVTH

KYGPPCPPCPAPEAAGGPSVFLFPP

QGLSSPVTKSFNRGEC

KPKDTLMISRTPEVTCVVVDVSQED

PEVQFNWYVDGVEVHNAKTKPREEQ

FNSTYRVVSVLTVLHQDWLNGKEYK

CKVSNKGLPSSIEKTISKAKGQPRE

PQVYTLPPSQEEMTKNQVSLTCLVK

GFYPSDIAVEWESNGQPENNYKTTP

PVLDSDGSFFLYSRLTVDKSRWQEG

NVFSCSVMHEALHNHYTQKSLSLSL

GK

PSMB127
EVQLLESGGGLVQPGGSLRLSCAAS
138
EIVLTQSPATLSLSPGERATLS
139

(FAB
GFTFKSDAMHWVRQAPGKGLEWVSE

CRASQSVSSYLAWYQQKPGQAP

PSMB83)
ISGSGGYTNYADSVKGRFTISRDNS

RLLIYDASNRATGIPARFSGSG

KNTLYLQMNSLRAEDTAVYYCARDS

SGTDFTLTISSLEPEDFAVYYC

YDSSLYVGDYFDYWGQGTLVTVSSA

QQRSNWPLTFGQGTKVEIKRTV

STKGPSVFPLAPCSRSTSESTAALG

AAPSVFIFPPSDEQLKSGTASV

CLVKDYFPEPVTVSWNSGALTSGVH

VCLLNNFYPREAKVQWKVDNAL

TFPAVLQSSGLYSLSSVVTVPSSSL

QSGNSQESVTEQDSKDSTYSLS

GTKTYTCNVDHKPSNTKVDKRVESK

STLTLSKADYEKHKVYACEVTH

YGPPCPPCPAPEAAGGPSVFLFPPK

QGLSSPVTKSFNRGEC

PKDTLMISRTPEVTCVVVDVSQEDP

EVQFNWYVDGVEVHNAKTKPREEQF

NSTYRVVSVLTVLHQDWLNGKEYKC

KVSNKGLPSSIEKTISKAKGQPREP

QVYTLPPSQEEMTKNQVSLTCLVKG

FYPSDIAVEWESNGQPENNYKTTPP

VLDSDGSFFLYSRLTVDKSRWQEGN

VFSCSVMHEALHNHYTQKSLSLSLG

K

PSMB126
QVQLVQSGAEVKKPGSSVKVSCKAS
140
DIQMTQSPSSLSASVGDRVTIT
137

(FAB
GGTFDDYAISWVRQAPGQGLEWMGR

CRASQSISSYLNWYQQKPGKAP

PSMB80)
IDPIEGTANYAQKFQGRVTITADES

KLLIYAASSLQSGVPSRFSGSG

TSTAYMELSSLRSEDTAVYYCARDR

SGTDFTLTISSLQPEDFATYYC

YYYDGVYWYSDYFDYWGQGTLVTVS

QQSYSTPLTFGQGTKVEIKRTV

SASTKGPSVFPLAPCSRSTSESTAA

AAPSVFIFPPSDEQLKSGTASV

LGCLVKDYFPEPVTVSWNSGALTSG

VCLLNNFYPREAKVQWKVDNAL

VHTFPAVLQSSGLYSLSSVVTVPSS

QSGNSQESVTEQDSKDSTYSLS

SLGTKTYTCNVDHKPSNTKVDKRVE

STLTLSKADYEKHKVYACEVTH

SKYGPPCPPCPAPEAAGGPSVFLFP

QGLSSPVTKSFNRGEC

PKPKDTLMISRTPEVTCVVVDVSQE

DPEVQFNWYVDGVEVHNAKTKPREE

QFNSTYRVVSVLTVLHQDWLNGKEY

KCKVSNKGLPSSIEKTISKAKGQPR

EPQVYTLPPSQEEMTKNQVSLTCLV

KGFYPSDIAVEWESNGQPENNYKTT

PPVLDSDGSFFLYSRLTVDKSRWQE

GNVFSCSVMHEALHNHYTQKSLSLS

LGK

PSMB124
QVQLVQSGAEVKKPGSSVKVSCKAS
141
DIQMTQSPSSLSASVGDRVTIT
142

(FAB
GGTFSSYAISWVRQAPGQGLEWMGW

CRASQSISGWLNWYQQKPGKAP

PSMB56)
ISPYNGNANYAQKFQGRVTITADES

KLLIYAASSLQSGVPSRFSGSG

TSTAYMELSSLRSEDTAVYYCARDS

SGTDFTLTISSLQPEDFATYYC

DRSYNLDYWGQGTLVTVSSASTKGP

QQSYSTPLTFGQGTKVEIKRTV

SVFPLAPCSRSTSESTAALGCLVKD

AAPSVFIFPPSDEQLKSGTASV

YFPEPVTVSWNSGALTSGVHTFPAV

VCLLNNFYPREAKVQWKVDNAL

LQSSGLYSLSSVVTVPSSSLGTKTY

QSGNSQESVTEQDSKDSTYSLS

TCNVDHKPSNTKVDKRVESKYGPPC

STLTLSKADYEKHKVYACEVTH

PPCPAPEAAGGPSVFLFPPKPKDTL

QGLSSPVTKSFNRGEC

MISRTPEVTCVVVDVSQEDPEVQFN

WYVDGVEVHNAKTKPREEQFNSTYR

VVSVLTVLHQDWLNGKEYKCKVSNK

GLPSSIEKTISKAKGQPREPQVYTL

PPSQEEMTKNQVSLTCLVKGFYPSD

IAVEWESNGQPENNYKTTPPVLDSD

GSFFLYSRLTVDKSRWQEGNVFSCS

VMHEALHNHYTQKSLSLSLGK

PSMB123
EVQLVQSGAEVKKPGESLKISCKGS
143
EIVLTQSPATLSLSPGERATLS
144

(FAB
GYSFTSYWIGWVRQMPGKGLEWMGI

CRASQSVASDLAWYQQKPGQAP

PSMB55)
IYPGDSDTRYSPSFQGQVTISADKS

RLLIYFASNRATGIPARFSGSG

ISTAYLQWSSLKASDTAMYYCARGL

SGTDFTLTISSLEPEDFAVYYC

PIWYLDYWGQGTLVTVSSASTKGPS

QQSITWPFTFGQGTKVEIKRTV

VFPLAPCSRSTSESTAALGCLVKDY

AAPSVFIFPPSDEQLKSGTASV

FPEPVTVSWNSGALTSGVHTFPAVL

VCLLNNFYPREAKVQWKVDNAL

QSSGLYSLSSVVTVPSSSLGTKTYT

QSGNSQESVTEQDSKDSTYSLS

CNVDHKPSNTKVDKRVESKYGPPCP

STLTLSKADYEKHKVYACEVTH

PCPAPEAAGGPSVFLFPPKPKDTLM

QGLSSPVTKSFNRGEC

ISRTPEVTCVVVDVSQEDPEVQFNW

YVDGVEVHNAKTKPREEQFNSTYRV

VSVLTVLHQDWLNGKEYKCKVSNKG

LPSSIEKTISKAKGQPREPQVYTLP

PSQEEMTKNQVSLTCLVKGFYPSDI

AVEWESNGQPENNYKTTPPVLDSDG

SFFLYSRLTVDKSRWQEGNVFSCSV

MHEALHNHYTQKSLSLSLGK

PSMB122
QVQLVQSGAEVKKPGSSVKVSCKAS
145
DIQMTQSPSSLSASVGDRVTIT
133

(FAB
GGTFSSYAISWVRQAPGQGLEWMGW

CRASQSIDRWLNWYQQKPGKAP

PSMB51)
IIPYNGNANYAQKFQGRVTITADES

KLLIYAASSLQSGVPSRFSGSG

TSTAYMELSSLRSEDTAVYYCARVN

SGTDFTLTISSLQPEDFATYYC

SAALVWERLDYWGQGTLVTVSSAST

QQSPRYPLTFGQGTKVEIKRTV

KGPSVFPLAPCSRSTSESTAALGCL

AAPSVFIFPPSDEQLKSGTASV

VKDYFPEPVTVSWNSGALTSGVHTF

VCLLNNFYPREAKVQWKVDNAL

PAVLQSSGLYSLSSVVTVPSSSLGT

QSGNSQESVTEQDSKDSTYSLS

KTYTCNVDHKPSNTKVDKRVESKYG

STLTLSKADYEKHKVYACEVTH

PPCPPCPAPEAAGGPSVFLFPPKPK

QGLSSPVTKSFNRGEC

DTLMISRTPEVTCVVVDVSQEDPEV

QFNWYVDGVEVHNAKTKPREEQFNS

TYRVVSVLTVLHQDWLNGKEYKCKV

SNKGLPSSIEKTISKAKGQPREPQV

YTLPPSQEEMTKNQVSLTCLVKGFY

PSDIAVEWESNGQPENNYKTTPPVL

DSDGSFFLYSRLTVDKSRWQEGNVF

SCSVMHEALHNHYTQKSLSLSLGK

PSMB121
QVQLVQSGAEVKKPGSSVKVSCKAS
146
EIVLTQSPATLSLSPGERATLS
147

(FAB
GGTFSSYAISWVRQAPGQGLEWMGG

CRASQSVSKWLAWYQQKPGQAP

PSMB49)
IIPIFGTANYAQKFQGRVTITADES

RLLIYDASNRATGIPARFSGSG

TSTAYMELSSLRSEDTAVYYCARAS

SGTDFTLTISSLEPEDFAVYYC

RVWHASYGYLDYWGQGTLVTVSSAS

QQRFTAPWTFGQGTKVEIKRTV

TKGPSVFPLAPCSRSTSESTAALGC

AAPSVFIFPPSDEQLKSGTASV

LVKDYFPEPVTVSWNSGALTSGVHT

VCLLNNFYPREAKVQWKVDNAL

FPAVLQSSGLYSLSSVVTVPSSSLG

QSGNSQESVTEQDSKDSTYSLS

TKTYTCNVDHKPSNTKVDKRVESKY

STLTLSKADYEKHKVYACEVTH

GPPCPPCPAPEAAGGPSVFLFPPKP

QGLSSPVTKSFNRGEC

KDTLMISRTPEVTCVVVDVSQEDPE

VQFNWYVDGVEVHNAKTKPREEQFN

STYRVVSVLTVLHQDWLNGKEYKCK

VSNKGLPSSIEKTISKAKGQPREPQ

VYTLPPSQEEMTKNQVSLTCLVKGF

YPSDIAVEWESNGQPENNYKTTPPV

LDSDGSFFLYSRLTVDKSRWQEGNV

FSCSVMHEALHNHYTQKSLSLSLGK

PSMB120
EVQLVQSGAEVKKPGESLKISCKGS
148
DIVMTQSPDSLAVSLGERATIN
149

(FAB
GYSFTSYWIGWVRQMPGKGLEWMGI

CKSSQSVLYSSNNKNYLAWYQQ

PSMB25)
IYPGDSDTRYSPSFQGQVTISADKS

KPGQPPKLLIYWASTRESGVPD

ISTAYLQWSSLKASDTAMYYCARGW

RFSGSGSGTDFTLTISSLQAED

AYDRGLDYWGQGTLVTVSSASTKGP

VAVYYCQQYYSTPLTFGQGTKV

SVFPLAPCSRSTSESTAALGCLVKD

EIKRTVAAPSVFIFPPSDEQLK

YFPEPVTVSWNSGALTSGVHTFPAV

SGTASVVCLLNNFYPREAKVQW

LQSSGLYSLSSVVTVPSSSLGTKTY

KVDNALQSGNSQESVTEQDSKD

TCNVDHKPSNTKVDKRVESKYGPPC

STYSLSSTLTLSKADYEKHKVY

PPCPAPEAAGGPSVFLFPPKPKDTL

ACEVTHQGLSSPVTKSFNRGEC

MISRTPEVTCVVVDVSQEDPEVQFN

WYVDGVEVHNAKTKPREEQFNSTYR

VVSVLTVLHQDWLNGKEYKCKVSNK

GLPSSIEKTISKAKGQPREPQVYTL

PPSQEEMTKNQVSLTCLVKGFYPSD

IAVEWESNGQPENNYKTTPPVLDSD

GSFFLYSRLTVDKSRWQEGNVFSCS

VMHEALHNHYTQKSLSLSLGK

PSMB119
EVQLVQSGAEVKKPGESLKISCKGS
150
DIVMTQSPDSLAVSLGERATIN
149

(FAB
GYSFTSYWIGWVRQMPGKGLEWMGI

CKSSQSVLYSSNNKNYLAWYQQ

PSMB18)
IYPGDSDTRYSPSFQGQVTISADKS

KPGQPPKLLIYWASTRESGVPD

ISTAYLQWSSLKASDTAMYYCARAY

RFSGSGSGTDFTLTISSLQAED

HYSKGLDYWGQGTLVTVSSASTKGP

VAVYYCQQYYSTPLTFGQGTKV

SVFPLAPCSRSTSESTAALGCLVKD

EIKRTVAAPSVFIFPPSDEQLK

YFPEPVTVSWNSGALTSGVHTFPAV

SGTASVVCLLNNFYPREAKVQW

LQSSGLYSLSSVVTVPSSSLGTKTY

KVDNALQSGNSQESVTEQDSKD

TCNVDHKPSNTKVDKRVESKYGPPC

STYSLSSTLTLSKADYEKHKVY

PPCPAPEAAGGPSVFLFPPKPKDTL

ACEVTHQGLSSPVTKSFNRGEC

MISRTPEVTCVVVDVSQEDPEVQFN

WYVDGVEVHNAKTKPREEQFNSTYR

VVSVLTVLHQDWLNGKEYKCKVSNK

GLPSSIEKTISKAKGQPREPQVYTL

PPSQEEMTKNQVSLTCLVKGFYPSD

IAVEWESNGQPENNYKTTPPVLDSD

GSFFLYSRLTVDKSRWQEGNVFSCS

VMHEALHNHYTQKSLSLSLGK

PSMB87
EVQLVQSGAEVKKPGESLKISCKGS
151
DIQMTQSPSSLSASVGDRVTIT
137

(FAB
GYSFTSYWISWVRQMPGKGLEWMGI

CRASQSISSYLNWYQQKPGKAP

PSMB58)
IYPGDSYTRYSPSFQGQVTISADKS

KLLIYAASSLQSGVPSRFSGSG

ISTAYLQWSSLKASDTAMYYCARDY

SGTDFTLTISSLQPEDFATYYC

EWELFDSRLDYWGQGTLVTVSSAST

QQSYSTPLTFGQGTKVEIKRTV

KGPSVFPLAPCSRSTSESTAALGCL

AAPSVFIFPPSDEQLKSGTASV

VKDYFPEPVTVSWNSGALTSGVHTF

VCLLNNFYPREAKVQWKVDNAL

PAVLQSSGLYSLSSVVTVPSSSLGT

QSGNSQESVTEQDSKDSTYSLS

KTYTCNVDHKPSNTKVDKRVESKYG

STLTLSKADYEKHKVYACEVTH

PPCPPCPAPEAAGGPSVFLFPPKPK

QGLSSPVTKSFNRGEC

DTLMISRTPEVTCVVVDVSQEDPEV

QFNWYVDGVEVHNAKTKPREEQFNS

TYRVVSVLTVLHQDWLNGKEYKCKV

SNKGLPSSIEKTISKAKGQPREPQV

YTLPPSQEEMTKNQVSLTCLVKGFY

PSDIAVEWESNGQPENNYKTTPPVL

DSDGSFFLYSRLTVDKSRWQEGNVF

SCSVMHEALHNHYTQKSLSLSLGK

A monospecific anti-PSMA antibody PSMB119 was generated comprising the VH and VL regions having the VH of SEQ ID NO: 130 and the VL of SEQ ID NO: 131 and an IgG4 constant region with S228P, F234A, and L235A substitutions. A monospecific anti-PSMA antibody PSMB120 was generated comprising the VH and VL regions having the VH of SEQ ID NO: 128 and the VL of SEQ ID NO: 129 and an IgG4 constant region with S228P, F234A, and L235A substitutions. A monospecific anti-PSMA antibody PSMB121 was generated comprising the VH and VL regions having the VH of SEQ ID NO: 126 and the VL of SEQ ID NO: 127 and an IgG4 constant region with S228P, F234A, and L235A substitutions. A monospecific anti-PSMA antibody PSMB122 was generated comprising the VH and VL regions having the VH of SEQ ID NO: 125 and the VL of SEQ ID NO: 111 and an IgG4 constant region with S228P, F234A, and L235A substitutions. A monospecific anti-PSMA antibody PSMB123 was generated comprising the VH and VL regions having the VH of SEQ ID NO: 123 and the VL of SEQ ID NO: 124 and an IgG4 constant region with S228P, F234A, and L235A substitutions. A monospecific anti-PSMA antibody PSMB124 was generated comprising the VH and VL regions having the VH of SEQ ID NO: 121 and the VL of SEQ ID NO: 122 and an IgG4 constant region with S228P, F234A, and L235A substitutions. A monospecific anti-PSMA antibody PSMB126 was generated comprising the VH and VL regions having the VH of SEQ ID NO: 118 and the VL of SEQ ID NO: 119 and an IgG4 constant region with S228P, F234A, and L235A substitutions. A monospecific anti-PSMA antibody PSMB127 was generated comprising the VH and VL regions having the VH of SEQ ID NO: 116 and the VL of SEQ ID NO: 117 and an IgG4 constant region with S228P, F234A, and L235A substitutions. A monospecific anti-PSMA antibody PSMB128 was generated comprising the VH and VL regions having the VH of SEQ ID NO: 114 and the VL of SEQ ID NO: 115 and an IgG4 constant region with S228P, F234A, and L235A substitutions. A monospecific anti-PSMA antibody PSMB129 was generated comprising the VH and VL regions having the VH of SEQ ID NO: 110 and the VL of SEQ ID NO: 111 and an IgG4 constant region with S228P, F234A, and L235A substitutions. A monospecific anti-PSMA antibody PSMB130 was generated comprising the VH and VL regions having the VH of SEQ ID NO: 112 and the VL of SEQ ID NO: 113 and an IgG4 constant region with S228P, F234A, and L235A substitutions.

2-5 Crystal Structure of Human PSMA ECD Bound to Anti-PSMA Fab PSMB83 (Aka PSMM84)

PSMA is a homodimeric protein expressed on the cell surface. PSMA is a type II integral glycoprotein of 750 residues per monomer, comprised of a large ECD domain (705 residues) with peptidase activity, a single pass TM domain, and a short 19 residue intracellular domain. The crystal structure of the extracellular region (ECD) of human PSMA bound to the anti-PSMA Fab arm of bispecific antibody PS3B27 was determined to 3.15 Å resolution to better understand the combining site between PSMA and the antibody.

The extracellular region of human PSMA (residues 44-750) was expressed in High Five™ insect cells with an N-terminal gp67 signal peptide followed by a cleavable hexahistidine tag (SEQ ID NO: 596). The secreted protein was purified from supernatant by a three-step procedure comprising of an initial Ni²⁺-NTA affinity capture, TEV-mediated cleavage of the histidine tag followed by an inverse affinity chromatography step, and a final size-exclusion chromatography step. Purified PSMA-ECD was flash-frozen in liquid nitrogen and stored at −80° C. in 10 mM HEPES pH 7.4, 150 mM NaCl, 2 mM CaCl₂), 0.1 mM ZnCl2.

The Fab of PSMB83 (aka PSMM84), which is the parental anti-PSMA Fab arm in bispecific antibody PS3B27, was expressed in HEK293 Expi cells with a hexahistidine tag (SEQ ID NO: 596) and purified using affinity (HisTrap, GE Healthcare) and size-exclusion chromatography (SEC-300, Phenomenex Yarra). The Fab was stored at 4° C. in 50 mM NaCl, 20 mM Tris pH 7.4.

The human PSMA ECD/PSMB83 Fab complex was prepared by a three-step procedure. First, the Fab was buffer exchanged into 20 mM MES pH 6.0, 150 mM NaCl. Then, the Fab and PSMA were mixed (1.5 molar excess Fab over PSMA monomer) and incubated overnight at 4° C. while dialyzing into 20 mM IVIES pH 6.0. Finally, the complex was bound to a monoS 5/50 column in 20 mM MES pH 6.0 and eluted with a NaCl gradient.

Crystals suitable for X-ray diffraction were obtained using the sitting drop vapor-diffusion method at 20° C. and a Mosquito LCP robot (TTP Labtech). Crystals of PSMB83 Fab bound to human PSMA ECD were grown from 18% PEG 3 kDa, 0.2 M (NH₄)₂SO₄, 0.1 M Tris pH 8.5 with micro-seeds and the PSMA/Fab complex initially at 7.3 mg/mL. Crystals of free PSMB83 Fab were obtained from 25% PEG 3 kDa, 0.2 M LiCl, 0.1 M acetate pH 4.5 with the Fab initially at 8.8 mg/mL.

The structures were solved by molecular replacement (MR) with Phaser (Phaser Crystallographic Software, University of Cambridge). The MR search model for the PSMB83 (aka PSMM84) Fab structure was PDB code 4M60. The PSMA/Fab complex structure was solved using the crystal structures of PSMA (PDB code: 2C6G) and PSMB83 Fab (structure at 1.93 Å resolution; data not shown) as MR search models. The structures were refined with PHENIX (Adams, et al, 2004) and model adjustments were carried out using COOT (Emsley and Cowtan, 2004). All other crystallographic calculations were performed with the CCP4 suite of programs (Collaborative Computational Project Number 4, 1994). All molecular graphics were generated with PyMol (PyMOL Molecular Graphics System, Version 1.4.1, Schrödinger, LLC.) and complementarity determining regions (CDRs) were determined using the Kabat definition.

The PSMA/Fab structure includes Fab light chain residues 1-211, Fab heavy chain residues 1-224 (except for residues 138-146, which are disordered) and PSMA residues 56-750, which corresponds to the protease (residues 56-116 and 352-590), apical (residues 117-351) and helical (residues 591-750) domains, and seven of ten possible N-linked glycans (in Asn-76, -121, -140, -195, -459, -476, and -638) per PSMA dimer subunit. The PSMA active site is located at the interface between the three domains and it contains two zinc atoms coordinated by histidine (H377 and H553) and glutamate/aspartate (D387, catalytic E424, E425, and D453) residues and a water molecule. The crystal asymmetric unit contains one PSMA dimer with each subunit bound in a similar manner to a PSMB83 Fab. The Fab/PSMA combining site is well defined by the electron density map, which allows reliable positioning of the binding residues. The Fab and PSMA molecules are numbered sequentially in FIG. 25-FIG. 30.

The PSMB83 epitope, paratope and interactions. PSMB83, which is the parental anti-PSMA Fab arm in bispecific antibody PS3B27, recognizes a conformational and discontinuous epitope in the apical domain of PSMA (FIG. 25). The PSMA surface area buried by the Fab is around 700 Å². Specifically, the PSMB83 epitope residues are I138, F235, P237, G238, D244, Y299, Y300, Q303, K304, E307, and K324-P326. Helix a7 (residues Y299-E307) is a prevalent region of the epitope and binds across the Fab heavy and light chain CDRs. At one end of the helix, Y299 and Y300 form an aromatic cluster with Fab residues Y57^H, W94^Land PSMA residues F235 and P237, while E307, at the other helix end, forms a salt bridge with R91^Land hydrogen bonds Y32^L. FIG. 26 and FIG. 27 show the main interactions of PSMA with the PSMB83 light and heavy chains. The PSMB83 epitope residues are conserved between human and cynomolgus monkey (FIG. 28) and the bispecific antibody PS3B27 was demonstrated to bind with similar affinity to human and cyno PSMA. In contrast, the human to mouse G238A and, especially, Y300D epitope mutations are expected to lower PSMB83 binding affinity to mouse PSMA in comparison to human. The Y300D mutation disrupts a hydrogen bond contact with N59^Hand a π stacking interaction with W94^L.

The PSMB83 paratope is composed of residues from all CDRs except CDR-L2 and CDR-H1 (FIG. 29). Specifically, the paratope residues are light chain S30^L, Y32^L, R91^L, S92^L, W94^L, and heavy chain G56^H-N59^H, K65^H, G66^H, Y101^{, H}, V107^Hand D109^H. FIG. 30 shows the interaction contacts between PSMA and PSMB83. The accessible location of the epitope facilitates binding of the PSMB83 Fab arm in the PS3B27 bispecific antibody to membrane-bound PSMA, while the other Fab arm is still bound to CD3 in the T-cell membrane. PSMB83 is not expected to inhibit PSMA enzymatic activity since the antibody binds away from the active site and does not cause any significant structural changes in PSMA that could affect enzymatic function, such as loop movements that close the active site or displacement of catalytic residues (RMSD of 0.3 Å for Cα superposition of PSMA molecules in Fab bound and unbound (Barinka et al, 2007) structures).

2-6 Anti-PSMA Affinity Maturation

Affinity maturation was performed on anti-PSMA Fab phage clones from two PSMA affinity maturation libraries to identify an antibody with increased binding affinity compared to the parental PSMB127 (fab ID=PSMB83, aka PSMM84). Two libraries were generated for affinity maturation of PSMB127). In the first library heavy chain CDR1 and CDR2 were randomized according to the design in Table 24 (PH9H9L1). The H-CDR3 fragment was PCR amplified from pDR000024032 and digested with SacII+XhoI. This fragment was cloned into the PH9H9L1/PH9L3 library. This was transformed into E. coli MC1061F′ cells and phage was generated displaying this Fab library. In the second library light chain CDRs were randomized according to the design in Table 25 (PH9L3L3). The heavy chain from PSMB83 (PSMH360) was PCR amplified and digested with NcoI+XhoI. This fragment was cloned into the PH9L3L3 library DNA (ELN: De Novo 2010 phage library SRI-021). This was transformed into E. coli MC1061F′ cells and phage was generated displaying this Fab library.

TABLE 24

PH9H9L1 Library design

Position
Parent AA
Library AA

30
S
D, K, S

31
S
D, N, S, T

32
Y
A, D, S, Y

33
A
A, D, G, S, W, Y

35
S
H, N, S

50
A
A, E, L, N, R, T, W, Y

52
S
A, D, L, N, R, S

54
S
A, E, N, S, Y

57
S
D, N, R, S, T, Y

59
Y
E, G, N, Q, R, Y

TABLE 25

PH9L3L3 Library design

Position
Parent AA
Library AA

30
S
D, N, R, S

31
S
N, S, T

32
Y
D, N, R, S, Y

49
Y
E, H, K, Y

50
D
D, G, S, W, Y

53
N
D, N, S, T, Y

91
R
A, D, E, G, H, N, R, S, W, Y

92
S
A, D, E, G, H, N, R, S, W, Y

93
N
A, D, E, G, H, N, R, S, W, Y

94
W
A, D, E, G, H, N, R, S, W, Y

96
L
F, I, L, N, R, W, Y

A solution panning of the PSMA affinity maturation Fab-pIX libraries was performed against biotinylated human PSMA ECD for three rounds. The phage-bound antigen was captured on neutravidin beads (GE HealthCare Life Science Cat #78152104011150) according to the manufacturer's protocol, followed by extensive washes in 1×PBST (0.05% tween 20) and an hour-long incubation with unlabeled PSMA ECD in 500-fold molar excess of the biotinylated antigen. This panning yielded the clones, PSMXP46R3_59H09, PSMXP46R3_59H06, PSMXP46R3_59E03, PSMXP46R3_59C09, PSMXP46R3_59H01, PSMXP46R3_59F11, and PSMXP46R3_59F07.

To determine the expression level of the anti-PSMA fab clones, 96 well Maxisorb plates were coated overnight at 4 C with anti-human Fd IgG, washed, and blocked with 3% milk-PBS-0.05% Tween for 1 hour. The phage supernatant samples were serially diluted 2-fold for 11 dilutions in blocking buffer with the final well blank. 100 u of these solutions were captured on the coated plates for 1 hour. The plates were washed and 100 u of anti-F(ab′)2-HRP antibody was added for 1 hour. Plates were washed and developed with 100 u of peroxidase reagent and luminescence was read on the Envision (FIG. 31).

To determine the binding of the anti-PSMA fab clones to human and cynomolgus recombinant protein, 96 well Maxisorb plates were coated with 100 u of 5 ug/ml neutravidin overnight at 4 C. The plates were washed and blocked with 3% milk-PBS-0.05% Tween for 1 hour. Recombinant biotinylated human and cynomolgus PSMA proteins were captured at 2.5 ug/ml for 1 hour at room temperature. The plates were washed and 100 u of 2-fold serially diluted fab supernatant was capture for 1 hour at RT. The plate was washed and then there was a 2.5-hour incubation with 200 ul 0.3% milk in PBST to wash away some of the weak affinity fabs. Then there was another 30 minutes incubation with fresh 200 ul 0.3% milk in PBST to remove more weak affinity fabs. The plates were washed and 100 ul of anti-F(ab′)2-HRP antibody was added for 1 hour. Plates were washed and developed with 100 u of peroxidase reagent and luminescence was read on the Envision (FIG. 32 and FIG. 33).

FIG. 31 demonstrates that the protein expression of the parental Fab and affinity matured Fabs were similar. The y axis values represent the luminescence of the detection reagent which equates to the abundance of fab protein over the dilution curve; the higher the luminescence reading, the more protein in the well which decreased with successive two-fold dilutions. There was more protein in the wells with affinity mature fabs but the increase over the parental is at most five times greater as demonstrated by the EC50 values (which is the concentration of protein that gives half of the maximal response). These data demonstrate the difference in PSMA binding profiles in FIG. 32 and FIG. 33 is not due to a difference in Fab concentration.

FIG. 32 demonstrates improved binding of the affinity matured Fabs to the human recombinant antigen over the parental anti-PSMA Fab (PSMB83). Again, the y axis of the graph represents luminescence values. In this case the larger the value means more Fab bound to the human PSMA protein. This is a measure of binding as increased concentrations of Fab (along the x axis) generate higher luminescence values. There was negligible binding of the parental Fab under these conditions as demonstrated by the absence of signal even at high concentrations (open circles along the x axis). Binding of the affinity matured fabs was observed over the concentrations tested which equates to stronger binding capacity to the human PSMA protein. Given that parental Fab binding to human PSMA protein was zero, no EC50 could be generated.

FIG. 33 demonstrates improved binding of the affinity matured Fabs to the cynomolgus recombinant antigen over the parental anti-PSMA Fab (PSMB83). Again, the y axis of the graph represents luminescence values. In this case the larger the value means more Fab bound to the cynomolgus PSMA protein. This is a measure of binding as increased concentrations of Fab (along the x axis) generate higher luminescence values. There was negligible binding of the parental Fab under these conditions as demonstrated by the absence of signal even at high concentrations (open circles along the x axis). Binding of the affinity matured Fabs was observed over the concentrations tested which equates to stronger binding capacity to the human PSMA protein. Given that parental Fab binding to human PSMA protein was zero, no EC50 could be generated for direct comparison.

Overall, the phages' Fab binding profiles demonstrate improved binding to the human and cynomolgus recombinant antigen over the parental anti-PSMA mAb (PSMB127). This improvement is not a result of differences in Fab expression profiles, as demonstrated by FIG. 33 and FIG. 34, showing binding of affinity mature Fabs normalized to Fab expression levels. The top five Fab candidates identified from the ELISA screen were produced in monoclonal antibody format on IgG4 PAA. Table 26 lists the subsequent Mab identifiers and Tables 27 and 28 depict sequences for their variable regions and the sequences for the heavy and light chains, respectively.

TABLE 26

Top five affinity mature antibodies identified based on the ELISA

HC
LC

Well ID
SEQ ID
SEQ ID
MAB protein IDs

PSMXP46R3_59C09
PSMH859
PSML160
PSMB346

PSMXP46R3_59E03
PSMH859
PSML159
PSMB345

PSMXP46R3_59F07
PSMH862
PSML158
PSMB349

PSMXP46R3_59H01
PSMH860
PH9L3
PSMB347

PSMXP46R3_59H06
PSMH859
PH9L3
PSMB344

TABLE 27

VH and VL sequences of top five PSMA Fab candidates

MAB ID
VH Amino acid sequence
SEQ ID NO
VL Amino Acid Sequence
SEQ ID NO

PSMB344
EVQLLESGGGLVQPGGSLRLSCAASG
6
EIVLTQSPATLSLSPGERATLS
117

FTFKSDAMHWVRQAPGKGLEWVSEIS

CRASQSVSSYLAWYQQKPGQAP

GSGGYTNYADSMKGRFTISRDNSKNT

RLLIYDASNRATGIPARFSGSG

LYLQMNSLRAEDTAVYYCARDSYDSS

SGTDFTLTISSLEPEDFAVYYC

LYVGDYFDYWGQGTLVTVSS

QQRSNWPLTFGQGTKVEIK

PSMB345
EVQLLESGGGLVQPGGSLRLSCAASG
6
EIVLTQSPATLSLSPGERATLS
9

FTFKSDAMHWVRQAPGKGLEWVSEIS

CRASQSVSNYLAWYQQKPGQAP

GSGGYTNYADSMKGRFTISRDNSKNT

RLLIHDASNRATGIPARFSGSG

LYLQMNSLRAEDTAVYYCARDSYDSS

SGTDFTLTISSLEPEDFAVYYC

LYVGDYFDYWGQGTLVTVSS

QQRRNWPLTFGQGTKVEIK

PSMB346
EVQLLESGGGLVQPGGSLRLSCAASG
6
EIVLTQSPATLSLSPGERATLS
682

FTFKSDAMHWVRQAPGKGLEWVSEIS

CRASQSVSSYLAWYQQKPGQAP

GSGGYTNYADSMKGRFTISRDNSKNT

RLLIYDASYRATGIPARFSGSG

LYLQMNSLRAEDTAVYYCARDSYDSS

SGTDFTLTISSLEPEDFAVYYC

LYVGDYFDYWGQGTLVTVSS

QQRRNWPLTFGQGTKVEIK

PSMB347
EVQLLESGGGLVQPGGSLRLSCAASG
7
EIVLTQSPATLSLSPGERATLS
117

FTFKSDAMHWVRQAPGKGLEWVSEIS

CRASQSVSSYLAWYQQKPGQAP

GSGGYTNYADSMKSRFTISRDNSKNT

RLLIYDASNRATGIPARFSGSG

LYLQMNSLRAEDTAVYYCARDSYDSS

SGTDFTLTISSLEPEDFAVYYC

LYVGDYFDYWGQGTLVTVSS

QQRSNWPLTFGQGTKVEIK

PSMB349
EVQLLESGGGLVQPGGSLRLSCAASG
8
EIVLTQSPATLSLSPGERATLS
10

FTFKSDAMHWVRQAPGKGLEWVSEIS

CRASQSVSSYLAWYQQKPGQAP

GSGGYTNYADSLKGRFTISRDNSKNT

RLLIYDASNRATGIPARFSGSG

LYLQMNSLRAEDTAVYYCARDSYDSS

SGTDFTLTISSLEPEDFAVYYC

LYVGDYFDYWGQGTLVTVSS

QQRGNWPLTFGQGTKVEIK

TABLE 28

Heavy Chain and Light Chain sequences of top

five PSMA candidates in monoclonal antibody

format on IgG4 PAA

SEQ
Light Chain
SEQ

Heavy Chain Amino
ID
Amino Acid
ID

mAb ID
acid sequence
NO
Sequence
NO

PSMB344
EVQLLESGGGLVQPGGSLRLSCAA
11
EIVLTQSPATL
139

SGFTFKSDAMHWVRQAPGKGLEWV

SLSPGERATLS

SEISGSGGYTNYADSMKGRFTISR

CRASQSVSSYL

DNSKNTLYLQMNSLRAEDTAVYYC

AWYQQKPGQAP

ARDSYDSSLYVGDYFDYWGQGTLV

RLLIYDASNRA

TVSSASTKGPSVFPLAPCSRSTSE

TGIPARFSGSG

STAALGCLVKDYFPEPVTVSWNSG

SGTDFTLTISS

ALTSGVHTFPAVLQSSGLYSLSSV

LEPEDFAVYYC

VTVPSSSLGTKTYTCNVDHKPSNT

QQRSNWPLTFG

KVDKRVESKYGPPCPPCPAPEAAG

QGTKVEIKRTV

GPSVFLFPPKPKDTLMISRTPEVT

AAPSVFIFPPS

CVVVDVSQEDPEVQFNWYVDGVEV

DEQLKSGTASV

HNAKTKPREEQFNSTYRVVSVLTV

VCLLNNFYPRE

LHQDWLNGKEYKCKVSNKGLPSSI

AKVQWKVDNAL

EKTISKAKGQPREPQVYTLPPSQE

QSGNSQESVTE

EMTKNQVSLTCLVKGFYPSDIAVE

QDSKDSTYSLS

WESNGQPENNYKTTPPVLDSDGSF

STLTLSKADYE

FLYSRLTVDKSRWQEGNVFSCSVM

KHKVYACEVTH

HEALHNHYTQKSLSLSLGK

QGLSSPVTKSF

NRGEC

PSMB345
EVQLLESGGGLVQPGGSLRLSCAA
11
EIVLTQSPATL
14

SGFTFKSDAMHWVRQAPGKGLEWV

SLSPGERATLS

SEISGSGGYTNYADSMKGRFTISR

CRASQSVSNYL

DNSKNTLYLQMNSLRAEDTAVYYC

AWYQQKPGQAP

ARDSYDSSLYVGDYFDYWGQGTLV

RLLIHDASNRA

TVSSASTKGPSVFPLAPCSRSTSE

TGIPARFSGSG

STAALGCLVKDYFPEPVTVSWNSG

SGTDFTLTISS

ALTSGVHTFPAVLQSSGLYSLSSV

LEPEDFAVYYC

VTVPSSSLGTKTYTCNVDHKPSNT

QQRRNWPLTFG

KVDKRVESKYGPPCPPCPAPEAAG

QGTKVEIKRTV

GPSVFLEPPKPKDTLMISRTPEVT

AAPSVFIFPPS

CVVVDVSQEDPEVQFNWYVDGVEV

DEQLKSGTASV

HNAKTKPREEQFNSTYRVVSVLTV

VCLLNNFYPRE

LHQDWLNGKEYKCKVSNKGLPSSI

AKVQWKVDNAL

EKTISKAKGQPREPQVYTLPPSQE

QSGNSQESVTE

EMTKNQVSLTCLVKGFYPSDIAVE

QDSKDSTYSLS

WESNGQPENNYKTTPPVLDSDGSF

STLTLSKADYE

FLYSRLTVDKSRWQEGNVFSCSVM

KHKVYACEVTH

HEALHNHYTQKSLSLSLGK

QGLSSPVTKSF

NRGEC

PSMB346
EVQLLESGGGLVQPGGSLRLSC
11
EIVLTQSPATL
680

AASGFTFKSDAMHWVRQAPGKG

SLSPGERATLS

LEWVSEISGSGGYTNYADSMKG

CRASQSVSSYL

RFTISRDNSKNTLYLQMNSLRA

AWYQQKPGQAP

EDTAVYYCARDSYDSSLYVGDY

RLLIYDASYRA

FDYWGQGTLVTVSSASTKGPSV

TGIPARFSGSG

FPLAPCSRSTSESTAALGCLVK

SGTDFTLTISS

DYFPEPVTVSWNSGALTSGVHT

LEPEDFAVYYC

FPAVLQSSGLYSLSSVVTVPSS

QQRRNWPLTFG

SLGTKTYTCNVDHKPSNTKVDK

QGTKVEIKRTV

RVESKYGPPCPPCPAPEAAGGP

AAPSVFIFPPS

SVFLFPPKPKDTLMISRIPEVT

DEQLKSGTASV

CVVVDVSQEDPEVQFNWYVDGV

VCLLNNFYPRE

EVHNAKTKPREEQFNSTYRVVS

AKVQWKVDNAL

VLTVLHQDWLNGKEYKCKVSNK

QSGNSQESVTE

GLPSSIEKTISKAKGQPREPQV

QDSKDSTYSLS

YTLPPSQEEMTKNQVSLTCLVK

STLTLSKADYE

GFYPSDIAVEWESNGQPENNYK

KHKVYACEVTH

TTPPVLDSDGSFFLYSRLTVDK

QGLSSPVTKSF

SRWQEGNVFSCSVMHEALHNHY

NRGEC

TQKSLSLSLGK

PSMB347
EVQLLESGGGLVQPGGSLRLSC
12
EIVLTQSPATL
139

AASGFTFKSDAMHWVRQAPGKG

SLSPGERATLS

LEWVSEISGSGGYTNYADSMKS

CRASQSVSSYL

RFTISRDNSKNTLYLQMNSLRA

AWYQQKPGQAP

EDTAVYYCARDSYDSSLYVGDY

RLLIYDASNRA

FDYWGQGTLVTVSSASTKGPSV

TGIPARFSGSG

FPLAPCSRSTSESTAALGCLVK

SGTDFTLTISS

DYFPEPVTVSWNSGALTSGVHT

LEPEDFAVYYC

FPAVLQSSGLYSLSSVVTVPSS

QQRSNWPLTFG

SLGTKTYTCNVDHKPSNTKVDK

QGTKVEIKRTV

RVESKYGPPCPPCPAPEAAGGP

AAPSVFIFPPS

SVFLFPPKPKDTLMISRIPEVT

DEQLKSGTASV

CVVVDVSQEDPEVQFNWYVDGV

VCLLNNFYPRE

EVHNAKTKPREEQFNSTYRVVS

AKVQWKVDNAL

VLTVLHQDWLNGKEYKCKVSNK

QSGNSQESVTE

GLPSSIEKTISKAKGQPREPQV

QDSKDSTYSLS

YTLPPSQEEMTKNQVSLTCLVK

STLTLSKADYE

GFYPSDIAVEWESNGQPENNYK

KHKVYACEVTH

TTPPVLDSDGSFFLYSRLTVDK

QGLSSPVTKSF

SRWQEGNVFSCSVMHEALHNHY

NRGEC

TQKSLSLSLGK

PSMB349
EVQLLESGGGLVQPGGSLRLSC
13
EIVLTQSPATL
15

AASGFTFKSDAMHWVRQAPGKG

SLSPGERATLS

LEWVSEISGSGGYTNYADSLKG

CRASQSVSSYL

RFTISRDNSKNTLYLQMNSLRA

AWYQQKPGQAP

EDTAVYYCARDSYDSSLYVGDY

RLLIYDASNRA

FDYWGQGTLVTVSSASTKGPSV

TGIPARFSGSG

FPLAPCSRSTSESTAALGCLVK

SGTDFTLTISS

DYFPEPVTVSWNSGALTSGVHT

LEPEDFAVYYC

FPAVLQSSGLYSLSSVVTVPSS

QQRGNWPLTFG

SLGTKTYTCNVDHKPSNTKVDK

QGTKVEIKRTV

RVESKYGPPCPPCPAPEAAGGP

AAPSVFIFPPS

SVFLFPPKPKDTLMISRIPEVT

DEQLKSGTASV

CVVVDVSQEDPEVQFNWYVDGV

VCLLNNFYPRE

EVHNAKTKPREEQFNSTYRVVS

AKVQWKVDNAL

VLTVLHQDWLNGKEYKCKVSNK

QSGNSQESVTE

GLPSSIEKTISKAKGQPREPQV

QDSKDSTYSLS

YTLPPSQEEMTKNQVSLTCLVK

STLTLSKADYE

GFYPSDIAVEWESNGQPENNYK

KHKVYACEVTH

TTPPVLDSDGSFFLYSRLTVDK

QGLSSPVTKSF

SRWQEGNVFSCSVMHEALHNHY

NRGEC

TQKSLSLSLGK

The 3 different HC and 4 different LC were combined in a matrix format to expand the diversity of hits (Table 29). Given that the methionine in the CDR2 of PSMH860 is a posttranslational risk, a new sequence was generated with M64L and identified as PSMH865. PSMH865 was paired with PSML160 to generate Mab PSMB365.

TABLE 29

Matrix format of the 3 heavy chains and 4 light chains combined

PSMH859
PSMH860
PSMH862

PH9L3
PSMB344
PSMB347
PSMB358

PSML158
—
PSMB361
PSMB349

PSML159
PSMB345
PSMB362
PSMB359

PSML160
PSMB346
PSMB363
PSMB360

Tables 30 and 31 depict the sequences for the matrix recombined variable regions and the sequences for the heavy and light chains, respectively.

TABLE 30

VH and VL sequences of matrix recombined

PSMA hits

SEQ

SEQ

VH Amino acid
ID
VL Amino Acid
ID

MAB ID
sequence
NO
Sequence
NO

PSMB358
EVQLLESGGGLVQPGG
8
EIVLTQSPATLSLSPGE
117

SLRLSCAASGFTFKSD

RATLSCRASQSVSSYLA

AMHWVRQAPGKGLEWV

WYQQKPGQAPRLLIYDA

SEISGSGGYTNYADSL

SNRATGIPARFSGSGSG

KGRFTISRDNSKNTLY

TDFTLTISSLEPEDFAV

LQMNSLRAEDTAVYYC

YYCQQRSNWPLTFGQGT

ARDSYDSSLYVGDYFD

KVEIK

YWGQGTLVTVSS

PSMB359
EVQLLESGGGLVQPGG
8
EIVLTQSPATLSLSPGE
9

SLRLSCAASGFTFKSD

RATLSCRASQSVSNYLA

AMHWVRQAPGKGLEWV

WYQQKPGQAPRLLIHDA

SEISGSGGYTNYADSL

SNRATGIPARFSGSGSG

KGRFTISRDNSKNTLY

TDFTLTISSLEPEDFAV

LQMNSLRAEDTAVYYC

YYCQQRRNWPLTFGQGT

ARDSYDSSLYVGDYFD

KVEIK

YWGQGTLVTVSS

PSMB360
EVQLLESGGGLVQPGG
8
EIVLTQSPATLSLSPGE
682

SLRLSCAASGFTFKSD

RATLSCRASQSVSSYLA

AMHWVRQAPGKGLEWV

WYQQKPGQAPRLLIYDA

SEISGSGGYTNYADSL

SYRATGIPARFSGSGSG

KGRFTISRDNSKNTLY

TDFTLTISSLEPEDFAV

LQMNSLRAEDTAVYYC

YYCQQRRNWPLTFGQGT

ARDSYDSSLYVGDYFD

KVEIK

YWGQGTLVTVSS

PSMB361
EVQLLESGGGLVQPGG
7
EIVLTQSPATLSLSPGE
10

SLRLSCAASGFTFKSD

RATLSCRASQSVSSYLA

AMHWVRQAPGKGLEWV

WYQQKPGQAPRLLIYDA

SEISGSGGYTNYADSM

SNRATGIPARFSGSGSG

KSRFTISRDNSKNTLY

TDFTLTISSLEPEDFAV

LQMNSLRAEDTAVYYC

YYCQQRGNWPLTFGQGT

ARDSYDSSLYVGDYFD

KVEIK

YWGQGTLVTVSS

PSMB362
EVQLLESGGGLVQPGG
7
EIVLTQSPATLSLSPGE
9

SLRLSCAASGFTFKSD

RATLSCRASQSVSNYLA

AMHWVRQAPGKGLEWV

WYQQKPGQAPRLLIHDA

SEISGSGGYTNYADSM

SNRATGIPARFSGSGSG

KSRFTISRDNSKNTLY

TDFTLTISSLEPEDFAV

LQMNSLRAEDTAVYYC

YYCQQRRNWPLTFGQGT

ARDSYDSSLYVGDYFD

KVEIK

YWGQGTLVTVSS

PSMB363
EVQLLESGGGLVQPGG
7
EIVLTQSPATLSLSPGE
682

SLRLSCAASGFTFKSD

RATLSCRASQSVSSYLA

AMHWVRQAPGKGLEWV

WYQQKPGQAPRLLIYDA

SEISGSGGYTNYADSM

SYRATGIPARFSGSGSG

KSRFTISRDNSKNTLY

TDFTLTISSLEPEDFAV

LQMNSLRAEDTAVYYC

YYCQQRRNWPLTFGQGT

ARDSYDSSLYVGDYFD

KVEIK

YWGQGTLVTVSS

PSMB365
EVQLLESGGGLVQPGG
681
EIVLTQSPATLSLSPGE
682

SLRLSCAASGFTFKSD

RATLSCRASQSVSSYLA

AMHWVRQAPGKGLEWV

WYQQKPGQAPRLLIYDA

SEISGSGGYTNYADSL

SYRATGIPARFSGSGSG

KSRFTISRDNSKNTLY

TDFTLTISSLEPEDFAV

LQMNSLRAEDTAVYYC

YYCQQRRNWPLTFGQGT

ARDSYDSSLYVGDYFD

KVEIK

YWGQGTLVTVSS

TABLE 31

Heavy Chain and Light Chain sequences of matrix

recombined PSMA hits

Heavy Chain
SEQ
Light Chain
SEQ

Amino acid
ID
Amino Acid
ID

MAB ID
sequence
NO
Sequence
NO

PSMB358
EVQLLESGGGLVQPGG
13
EIVLTQSPATLSLSPGE
139

SLRLSCAASGFTFKSD

RATLSCRASQSVSSYLA

AMHWVRQAPGKGLEWV

WYQQKPGQAPRLLIYDA

SEISGSGGYTNYADSL

SNRATGIPARFSGSGSG

KGRFTISRDNSKNTLY

TDFTLTISSLEPEDFAV

LQMNSLRAEDTAVYYC

YYCQQRSNWPLTFGQGT

ARDSYDSSLYVGDYFD

KVEIKRTVAAPSVFIFP

YWGQGTLVTVSSASTK

PSDEQLKSGTASVVCLL

GPSVFPLAPCSRSTSE

NNFYPREAKVQWKVDNA

STAALGCLVKDYFPEP

LQSGNSQESVTEQDSKD

VTVSWNSGALTSGVHT

STYSLSSTLTLSKADYE

FPAVLQSSGLYSLSSV

KHKVYACEVTHQGLSSP

VTVPSSSLGTKTYTCN

VTKSFNRGEC

VDHKPSNTKVDKRVES

KYGPPCPPCPAPEAAG

GPSVFLFPPKPKDTLM

ISRTPEVTCVVVDVSQ

EDPEVQFNWYVDGVEV

HNAKTKPREEQFNSTY

RVVSVLTVLHQDWLNG

KEYKCKVSNKGLPSSI

EKTISKAKGQPREPQV

YTLPPSQEEMTKNQVS

LTCLVKGFYPSDIAVE

WESNGQPENNYKTTPP

VLDSDGSFFLYSRLTV

DKSRWQEGNVFSCSVM

HEALHNHYTQKSLSLS

LGK

PSMB359
EVQLLESGGGLVQPGG
13
EIVLTQSPATLSLSPGE
14

SLRLSCAASGFTFKSD

RATLSCRASQSVSNYLA

AMHWVRQAPGKGLEWV

WYQQKPGQAPRLLIHDA

SEISGSGGYTNYADSL

SNRATGIPARFSGSGSG

KGRFTISRDNSKNTLY

TDFTLTISSLEPEDFAV

LQMNSLRAEDTAVYYC

YYCQQRRNWPLTFGQGT

ARDSYDSSLYVGDYFD

KVEIKRTVAAPSVFIFP

YWGQGTLVTVSSASTK

PSDEQLKSGTASVVCLL

GPSVFPLAPCSRSTSE

NNFYPREAKVQWKVDNA

STAALGCLVKDYFPEP

LQSGNSQESVTEQDSKD

VTVSWNSGALTSGVHT

STYSLSSTLTLSKADYE

FPAVLQSSGLYSLSSV

KHKVYACEVTHQGLSSP

VTVPSSSLGTKTYTCN

VTKSFNRGEC

VDHKPSNTKVDKRVES

KYGPPCPPCPAPEAAG

GPSVFLFPPKPKDTLM

ISRTPEVTCVVVDVSQ

EDPEVQFNWYVDGVEV

HNAKTKPREEQFNSTY

RVVSVLTVLHQDWLNG

KEYKCKVSNKGLPSSI

EKTISKAKGQPREPQV

YTLPPSQEEMTKNQVS

LTCLVKGFYPSDIAVE

WESNGQPENNYKTTPP

VLDSDGSFFLYSRLTV

DKSRWQEGNVFSCSVM

HEALHNHYTQKSLSLS

LGK

PSMB360
EVQLLESGGGLVQPGG
13
EIVLTQSPATLSLSPGE
680

SLRLSCAASGFTFKSD

RATLSCRASQSVSSYLA

AMHWVRQAPGKGLEWV

WYQQKPGQAPRLLIYDA

SEISGSGGYTNYADSL

SYRATGIPARFSGSGSG

KGRFTISRDNSKNTLY

TDFTLTISSLEPEDFAV

LQMNSLRAEDTAVYYC

YYCQQRRNWPLTFGQGT

ARDSYDSSLYVGDYFD

KVEIKRTVAAPSVFIFP

YWGQGTLVTVSSASTK

PSDEQLKSGTASVVCLL

GPSVFPLAPCSRSTSE

NNFYPREAKVQWKVDNA

STAALGCLVKDYFPEP

LQSGNSQESVTEQDSKD

VTVSWNSGALTSGVHT

STYSLSSTLTLSKADYE

FPAVLQSSGLYSLSSV

KHKVYACEVTHQGLSSP

VTVPSSSLGTKTYTCN

VTKSFNRGEC

VDHKPSNTKVDKRVES

KYGPPCPPCPAPEAAG

GPSVFLFPPKPKDTLM

ISRTPEVTCVVVDVSQ

EDPEVQFNWYVDGVEV

HNAKTKPREEQFNSTY

RVVSVLTVLHQDWLNG

KEYKCKVSNKGLPSSI

EKTISKAKGQPREPQV

YTLPPSQEEMTKNQVS

LTCLVKGFYPSDIAVE

WESNGQPENNYKTTPP

VLDSDGSFFLYSRLTV

DKSRWQEGNVFSCSVM

HEALHNHYTQKSLSLS

LGK

PSMB361
EVQLLESGGGLVQPGG
12
EIVLTQSPATLSLSPGE
15

SLRLSCAASGFTFKSD

RATLSCRASQSVSSYLA

AMHWVRQAPGKGLEWV

WYQQKPGQAPRLLIYDA

SEISGSGGYTNYADSM

SNRATGIPARFSGSGSG

KSRFTISRDNSKNTLY

TDFTLTISSLEPEDFAV

LQMNSLRAEDTAVYYC

YYCQQRGNWPLTFGQGT

ARDSYDSSLYVGDYFD

KVEIKRTVAAPSVFIFP

YWGQGTLVTVSSASTK

PSDEQLKSGTASVVCLL

GPSVFPLAPCSRSTSE

NNFYPREAKVQWKVDNA

STAALGCLVKDYFPEP

LQSGNSQESVTEQDSKD

VTVSWNSGALTSGVHT

STYSLSSTLTLSKADYE

FPAVLQSSGLYSLSSV

KHKVYACEVTHQGLSSP

VTVPSSSLGTKTYTCN

VTKSFNRGEC

VDHKPSNTKVDKRVES

KYGPPCPPCPAPEAAG

GPSVFLFPPKPKDTLM

ISRTPEVTCVVVDVSQ

EDPEVQFNWYVDGVEV

HNAKTKPREEQFNSTY

RVVSVLTVLHQDWLNG

KEYKCKVSNKGLPSSI

EKTISKAKGQPREPQV

YTLPPSQEEMTKNQVS

LTCLVKGFYPSDIAVE

WESNGQPENNYKTTPP

VLDSDGSFFLYSRLTV

DKSRWQEGNVFSCSVM

HEALHNHYTQKSLSLS

LGK

PSMB362
EVQLLESGGGLVQPGG
12
EIVLTQSPATLSLSPGE
14

SLRLSCAASGFTFKSD

RATLSCRASQSVSNYLA

AMHWVRQAPGKGLEWV

WYQQKPGQAPRLLIHDA

SEISGSGGYTNYADSM

SNRATGIPARFSGSGSG

KSRFTISRDNSKNTLY

TDFTLTISSLEPEDFAV

LQMNSLRAEDTAVYYC

YYCQQRRNWPLTFGQGT

ARDSYDSSLYVGDYFD

KVEIKRTVAAPSVFIFP

YWGQGTLVTVSSASTK

PSDEQLKSGTASVVCLL

GPSVFPLAPCSRSTSE

NNFYPREAKVQWKVDNA

STAALGCLVKDYFPEP

LQSGNSQESVTEQDSKD

VTVSWNSGALTSGVHT

STYSLSSTLTLSKADYE

FPAVLQSSGLYSLSSV

KHKVYACEVTHQGLSSP

VTVPSSSLGTKTYTCN

VTKSFNRGEC

VDHKPSNTKVDKRVES

KYGPPCPPCPAPEAAG

GPSVFLFPPKPKDTLM

ISRTPEVTCVVVDVSQ

EDPEVQFNWYVDGVEV

HNAKTKPREEQFNSTY

RVVSVLTVLHQDWLNG

KEYKCKVSNKGLPSSI

EKTISKAKGQPREPQV

YTLPPSQEEMTKNQVS

LTCLVKGFYPSDIAVE

WESNGQPENNYKTTPP

VLDSDGSFFLYSRLTV

DKSRWQEGNVFSCSVM

HEALHNHYTQKSLSLS

LGK

PSMB363
EVQLLESGGGLVQPGG
12
EIVLTQSPATLSLSPGE
680

SLRLSCAASGFTFKSD

RATLSCRASQSVSSYLA

AMHWVRQAPGKGLEWV

WYQQKPGQAPRLLIYDA

SEISGSGGYTNYADSM

SYRATGIPARFSGSGSG

KSRFTISRDNSKNTLY

TDFTLTISSLEPEDFAV

LQMNSLRAEDTAVYYC

YYCQQRRNWPLTFGQGT

ARDSYDSSLYVGDYFD

KVEIKRTVAAPSVFIFP

YWGQGTLVTVSSASTK

PSDEQLKSGTASVVCLL

GPSVFPLAPCSRSTSE

NNFYPREAKVQWKVDNA

STAALGCLVKDYFPEP

LQSGNSQESVTEQDSKD

VTVSWNSGALTSGVHT

STYSLSSTLTLSKADYE

FPAVLQSSGLYSLSSV

KHKVYACEVTHQGLSSP

VTVPSSSLGTKTYTCN

VTKSFNRGEC

VDHKPSNTKVDKRVES

KYGPPCPPCPAPEAAG

GPSVFLFPPKPKDTLM

ISRTPEVTCVVVDVSQ

EDPEVQFNWYVDGVEV

HNAKTKPREEQFNSTY

RVVSVLTVLHQDWLNG

KEYKCKVSNKGLPSSI

EKTISKAKGQPREPQV

YTLPPSQEEMTKNQVS

LTCLVKGFYPSDIAVE

WESNGQPENNYKTTPP

VLDSDGSFFLYSRLTV

DKSRWQEGNVFSCSVM

HEALHNHYTQKSLSLS

LGK

PSMB365
EVQLLESGGGLVQPGG
679
EIVLTQSPATLSLSPGE
631

SLRLSCAASGFTFKSD

RATLSCRASQSVSSYLA

AMHWVRQAPGKGLEWV

WYQQKPGQAPRLLIYDA

SEISGSGGYTNYADSL

SYRATGIPARFSGSGSG

KSRFTISRDNSKNTLY

TDFTLTISSLEPEDFAV

LQMNSLRAEDTAVYYC

YYCQQRRNWPLTFGQGT

ARDSYDSSLYVGDYFD

KVEIKRTVAAPSVFIFP

YWGQGTLVTVSSASTK

PSDEQLKSGTASVVCLL

GPSVFPLAPCSRSTSE

NNFYPREAKVQWKVDNA

STAALGCLVKDYFPEP

LQSGNSQESVTEQDSKD

VTVSWNSGALTSGVHT

STYSLSSTLTLSKADYE

FPAVLQSSGLYSLSSV

KHKVYACEVTHQGLSSP

VTVPSSSLGTKTYTCN

VTKSFNRGEC

VDHKPSNTKVDKRVES

KYGPPCPPCPAPEAAG

GPSVFLFPPKPKDTLM

ISRTPEVTCVVVDVSQ

EDPEVQFNWYVDGVEV

HNAKTKPREEQFNSTY

RVVSVLTVLHQDWLNG

KEYKCKVSNKGLPSSI

EKTISKAKGQPREPQV

YTLPPSQEEMTKNQVS

LTCLVKGFYPSDIAVE

WESNGQPENNYKTTPP

VLDSDGSFFLYSRLTV

DKSRWQEGNVFSCSVM

HEALHNHYTQKSLSLS

LGK

Table 32 shows the CDR sequences for all affinity-matured PSMA hits.

TABLE 32

CDR sequences of affinity-matured PSMA hits

CDRs (SEQ ID NO:)

MAB ID

CDR1
CDR2
CDR3

PSMB344
HC
SDAMH
EISGSGGYT
DSYDSSLYV

(78)
NYADSMKG
GDYFDY

(1)
(80)

LC
RASQSVSSYLA
DASNRAT
QQRSNWPLT

(81)
(82)
(83)

PSMB345
HC
SDAMH
EISGSGGYT
DSYDSSLYV

(78)
NYADSMKG
GDYFDY

(1)
(80)

LC
RASQSVSNYLA
DASNRAT
QQRRNWPLT

(4)
(82)
(686)

PSMB346
HC
SDAMH
EISGSGGYT
DSYDSSLYV

(78)
NYADSMKG
GDYFDY

(1)
(80)

LC
RASQSVSSYLA
DASYRAT
QQRRNWPLT

(81)
(684)
(686)

PSMB347
HC
SDAMH
EISGSGGYT
DSYDSSLYV

(78)
NYADSMKS
GDYFDY

(2)
(80)

LC
RASQSVSSYLA
DASNRAT
QQRSNWPLT

(81)
(82)
(83)

PSMB349
HC
SDAMH
EISGSGGYT
DSYDSSLYV

(78)
NYADSLKG
GDYFDY

(3)
(80)

LC
RASQSVSSYLA
DASNRAT
QQRGNWPLT

(81)
(82)
(5)

PSMB358
HC
SDAMH
EISGSGGYT
DSYDSSLYV

(78)
NYADSLKG
GDYFDY

(3)
(80)

LC
RASQSVSSYLA
DASNRAT
QQRSNWPLT

(81)
(82)
(83)

PSMB359
HC
SDAMH
EISGSGGYT
DSYDSSLYV

(78)
NYADSLKG
GDYFDY

(3)
(80)

LC
RASQSVSNYLA
DASNRAT
QQRRNWPLT

(4)
(82)
(686)

PSMB360
HC
SDAMH
EISGSGGYT
DSYDSSLYV

(78)
NYADSLKG
GDYFDY

(3)
(80)

LC
RASQSVSSYLA
DASYRAT
QQRRNWPLT

(81)
(684)
(686)

PSMB361
HC
SDAMH
EISGSGGYT
DSYDSSLYV

(78)
NYADSMKS
GDYFDY

(2)
(632)

LC
RASQSVSSYLA
DASNRAT
QQRGNWPLT

(81)
(82)
(5)

PSMB362
HC
SDAMH
EISGSGGYT
DSYDSSLYV

(78)
NYADSMKS
GDYFDY

(2)
(80)

LC
RASQSVSNYLA
DASNRAT
QQRRNWPLT

(4)
(685)
(686)

PSMB363
HC
SDAMH
EISGSGGYT
DSYDSSLYV

(78)
NYADSMKS
GDYFDY

(2)
(80)

LC
RASQSVSSYLA
DASYRAT
QQRRNWPLT

(630)
(684)
(686)

PSMB365
HC
SDAMH
EISGSGGYT
DSYDSSLYV

(78)
NYADSLKS
GDYFDY

(683)
(80)

LC
RASQSVSSYLA
DASYRAT
QQRRNWPLT

(81)
(684)
(686)

3 Preparation and Functional Evaluation of PSMA×CD3 Bispecific Antibodies
3-1 Generation of PSMA×CD3 Bispecific Antibodies

Two types of affinity-matured PSMA×CD3 bispecific antibodies were generated: one specific for the targeting arm (e.g. affinity matured anti-PSMA) recombined with a high affinity CD3 arm CD3B376 (VH SEQ ID NO:652, VL SEQ ID NO:661; HC SEQ ID NO:640, LC SEQ ID NO:676) CD3 arm or low affinity CD3B450 (VH SEQ ID NO: 657, VL SEQ ID NO: 678, HC SEQ ID NO:675, LC SEQ ID NO:677) arm.

These parental mAbs are in the GenMab format (Labrijn et al, 2013) where the targeting parent (PSMA) contains the 409R GenMab mutation (native amino acid for IgG4), while the killing parent (CD3) contains the F405L GenMab mutation and R409K mutation. The monospecific anti-CD3 antibody was expressed as IgG4, having Fc substitutions S228P, F234A, L235A, F405L, and R409K (CD3 arm) (numbering according to EU index) in their Fc regions. The targeting parent (PSMA) is on human IgG4 with Fc substitutions S228P, F234A, L235A. The monospecific antibodies were expressed in HEK cell lines under CMV promoters.

The parental PSMA and CD3 antibodies were purified using a protein A column with an elution buffer of 100 mM NaAc pH3.5 and a neutralization buffer of 2.5M Tris, pH 7.2. The neutralized parental mAbs were used to make PSMA×CD3 bispecific antibodies. A portion of parental mabs were further buffer exchanged into D-PBS, pH 7.2 buffer for analytical measurements and assays.

Post purification, controlled Fab-arm exchange was performed to make bispecific antibodies. The parental PSMA antibodies were mixed with the desired parental CD3 antibody under reducing conditions in 75 mM-2-MEA (2-Mercaptoethylamine) and incubated at 31° C. for 4h or at room temperature overnight. The recombination reactions were based on molar ratios, where 6% excess PSMA parental mAbs was used to minimize the CD3 parental mAb remaining after recombination. The recombinations were subsequently dialyzed against 1×DPBS, pH 7.2 to remove the reductant.

The final bispecific antibodies produced, along with the parental mAbs (i.e. PSMA, CD3, or Null) used in the recombination reactions are listed in Table 33.

Selected PSMA hits were also paired with a non-killing arm (Null) to create negative controls for testing purposes. For control bispecific antibodies, B2M1, an RSV antibody in the IgG4 PAA format (VH SEQ ID NO:610, VL SEQ ID NO:611) was generated, purified and, combined with either the CD3 arm CD3B219-F405L, R409K to generate CD3B288 (CD3×null) or PSMA arms, PSMB122, PSMB126, PSMB130 to generate PS3B37, PS3B39 and PS3B40 respectively (PSMA×null). These PSMA specific affinity matured Mabs were crossed (as in methods above) to CD3B219 and CD3B376 to generate the bispecific antibodies shown in Table 32.

TABLE 33

Generation of affinity-matured PSMA × CD3 bispecific

antibodies generated from affinity matured PSMA hits

ID
Arm 1
HC
LC
Arm 2
HC
LC

PS3B60
PSMB344
PSMH859
PH9L3
CD3B219
CD3H141
CD3L66

PS3B61
PSMB345
PSMH859
PSML159
CD3B219
CD3H141
CD3L66

PS3B62
PSMB346
PSMH859
PSML160
CD3B219
CD3H141
CD3L66

PS3B63
PSMB347
PSMH860
PH9L3
CD3B219
CD3H141
CD3L66

PS3B64
PSMB349
PSMH862
PSML158
CD3B219
CD3H141
CD3L66

PS3B70
PSMB358
PSMH862
PH9L3
CD3B219
CD3H141
CD3L66

PS3B71
PSMB359
PSMH862
PSML159
CD3B219
CD3H141
CD3L66

PS3B72
PSMB360
PSMH862
PSML160
CD3B219
CD3H141
CD3L66

PS3B73
PSMB361
PSMH860
PSML158
CD3B219
CD3H141
CD3L66

PS3B74
PSMB362
PSMH860
PSML159
CD3B219
CD3H141
CD3L66

PS3B75
PSMB363
PSMH860
PSML160
CD3B219
CD3H141
CD3L66

PS3B76
PSMB358
PSMH862
PH9L3
CD3B376
CD3H219
CD3L150

PS3B77
PSMB349
PSMH862
PSML158
CD3B376
CD3H219
CD3L150

PS3B78
PSMB359
PSMH862
PSML159
CD3B376
CD3H219
CD3L150

PS3B79
PSMB360
PSMH862
PSML160
CD3B376
CD3H219
CD3L150

PS3B80
PSMB347
PSMH860
PH9L3
CD3B376
CD3H219
CD3L150

PS3B81
PSMB361
PSMH860
PSML158
CD3B376
CD3H219
CD3L150

PS3B82
PSMB362
PSMH860
PSML159
CD3B376
CD3H219
CD3L150

PS3B83
PSMB363
PSMH860
PSML160
CD3B376
CD3H219
CD3L150

PS3B84
PSMB344
PSMH859
PH9L3
CD3B376
CD3H219
CD3L150

PS3B85
PSMB345
PSMH859
PSML159
CD3B376
CD3H219
CD3L150

PS3B86
PSMB346
PSMH859
PSML160
CD3B376
CD3H219
CD3L150

PS3B90
PSMB365
PSMH865
PSML160
CD3B376
CD3H219
CD3L150

3-2 Evaluation of PSMA×CD3 Affinity Matured Bispecific Abs in LNCAP Cell Binding

PSMA×CD3 bispecific antibodies were tested for binding to PSMA positive cell line, LNCAP, to PSMA negative cell line, PC3. To assess the binding capabilities of the PSMA bispecific antibodies, the cell-binding assay was utilized (described previously). Bispecific antibodies were normalized for protein concentration and then incubated with the same number of cells expressing either human or cyno PSMA. The MFI at each concentration was collected by flow cytometry and plotted as a function of concentration. Data was transformed via log 10 and then plotted. Nonlinear regression of binding curves was done to determine EC50s.

These relative values were used for ranking PSMA binding to target cells. FIGS. 14-16 show LNCAP binding of all bispecific antibodies prepared. In FIG. 16, none of the constructs demonstrated binding to the PSMA negative cell line. In FIG. 14 and FIG. 15, all of the affinity matured hits demonstrated increased binding affinity through left shifted curves and increased cMax as compared to the parental Mab, PS2B27.

The interactions of the affinity matured bispecific antibodies with recombinant Cyno PSMA ECD and Human PSMA ECD were studied by Surface Plasmon Resonance (SPR) using a ProteOn XPR36 system (BioRad) as described previously for recombinant chimp PSMA ECD. All of the bispecific antibodies bind both targets with substantially the same affinity, KDs ranging from 0.05 nM to 0.27 nM for human PSMA ECD and from 0.05 nM to 0.23 nM from cyno PSMA ECD.

3-3 Evaluation of PSMA×CD3 Affinity Matured Bispecific Abs in Functional Cell Killing Assay

Based on the above data, affinity measurements, and sequence identity, three PSMA antibodies, PSMB347, PSMB360 and PSMB365 as bispecifics with either CD3B219 or CD3B376, were further characterized for the ability to mediate PSMA specific, redirected T cell cytotoxicity. T-cell mediated killing was measured using a caspase cytotoxicity assay, which indirectly measures cell killing via cleavage of a fluorescent substrate by active caspase 3/7. Cleavage of the substrate results in a fluorescent DNA dye, with fluorescence restricted to the cell nucleus. Repeated fluorescence measurements are taken in each well throughout the course of the assay, using a motorized 10× objective, capable of precisely imaging well(s) at the same coordinates. Target cell populations are identified based on defined size restrictions and/or through the use of a secondary label. Frozen Pan CD3+ T-cells (purchased from Biological Specialty Corporation, Colmar, PA) were isolated by negative selection from normal healthy donors. Prostate cancer cells, expressing PSMA, (LNCaP, C42) were cultured in RPMI 1640 with 10% HI FBS+supplements (purchased from Life Technologies).

T-cells and target cells were combined at an effector to target ratio (E:T) of 3:1 in Phenol Red free RPMI+10% FBS and supplements (Life Technologies), without selection reagents, and 0.6 uL of NucView caspase reagent (Essen Bioscience) was added to each mL of cells, per manufacturer guidelines. A total volume of 0.1 mL cells were added to appropriate wells of a clear, 96-well flat-bottom plate (BD Falcon). PS3B27 (CD3×PSMA), CD3B288 (CD3×Null) or

PS3B46 (PSMA×Null) Bispecific antibodies were prepared at 2× final concentration in Phenol Red free RPMI, prepared as indicated above, and 0.1 mL of compounds were added to each well. After 30 minute incubation at room temperature to minimize cell aggregation at the edge of wells, plates were transferred to the Zoom Incucyte instrument (Essen Bioscience). The Incucyte Instrument resides in a humidified incubator set at 37° C., 5% CO2.

Processing definitions on the Incucyte were designed for each cell line tested, per manufacture guidelines. Measurements were taken every six hours, until a plateau in the caspase signal was observed, and followed by three or more successive decreases from the maximum signal in the well(s) containing the highest concentration of the test compound(s). As the data shows in FIG. 17, the curves for PS3B80, PS3B79, PS3B89, PS3B90, PS3B63, and PS3B72 are left shifted indicating increased potency over PS3B27. The null arm controls did not induce cell death as expected.

3-4 Anti-Tumor Efficacy in Tumorigenesis Prevention of LnCaP Xenografts in Humanized NSG Mice

Efficacy of PS3B79 and PS3B90 was evaluated in established 3D LnCaP AR.TB human prostate cancer xenografts in male NOD.Cg-Prkdc^scidIl2rg^tm1Wjl/SzJ (NSG) mice humanized intraperitoneally (ip) with human T cells. PS3B79 and PS3B90 at 2.5 and 5 mg/kg or Null×CD3B376 antibody control was dosed q3d-q4d on Days 36, 39, 43, 47, 50, 53, 56, 60, and 63 for a total of 8 doses. On day 53 post-tumor implant, which was the last date of the study when nine (9) animals remained per group, tumor growth inhibition (% TGI) was calculated. Statistically significant tumor growth inhibition was observed for PS3B79 at 5 mg/kg with 42% TGI (Two-way ANOVA with Bonferroni test, *p<0.0001, FIG. 20), and for PS3B90 at 2.5 and 5 mg/kg with 53% and 33% respectively compared to Null×CD3 control (Two-way ANOVA with Bonferroni test, *p<0.001, FIG. 21). Thus, CD3B376 is able to induce T cell activation and cytotoxicity in vivo and result in tumor growth inhibition in a bispecific format with high affinity PSMA binding arms, PSMB360 and PSMB365.

4 Preparation and Functional Evaluation of IL1RAP×CD3 Bispecific Antibodies

IL1RAP monoclonal antibodies used in and suitable for use in the bispecific antibodies of the present disclosure are described in U.S. Patent Application Publication No. 20170121420A1, the disclosure of which is hereby incorporated by reference in its entirety. Fifteen monospecific IL1RAP antibodies (see Table 6 of US20170121420A1) were expressed as IgG4, having Fc substitutions S228P, L234A, and L235A. CD3 parental mAbs were expressed as IgG4 with S228P, L234A, L235A, F405L, and R409K (numbering according to EU index). A monospecific anti-CD3 antibody CD3B220 was also generated comprising the VH and VL regions having the VH of SEQ ID NO: 92 and the VL of SEQ ID NO: 93 and IgG4 constant region with S228P, L234A, L235A, F405L, and R409K substitutions.

The monospecific antibodies were purified using standard methods using a Protein A column. After elution, the pools were neutralized to pH 7 and dialyzed into 1×D-PBS, pH 7.2.

Bispecific IL1RAP×CD3 antibodies were generated by combining a monospecific CD3 mAb (CD3B376: VH of SEQ ID NO: 652 and VL of SEQ ID NO: 661; or CD3B450: VH of SEQ ID NO: 657 and VL of SEQ ID NO: 678) and a monospecific IL1RAP mAb through controlled Fab-arm exchange (as described in WO2011/131746). Briefly, at about 1-20 mg/mL at a molar ratio of 1.08:1 of anti-IL1RAP/anti-CD3 antibody in DPBS, pH 7-7.4 and 75 mM 2-mercaptoethanolamine (2-MEA) were mixed together and incubated at 25-37° C. for 2-6 hours, followed by removal of the 2-MEA via dialysis, diafiltration, tangential flow filtration to remove the reductant and enable bispecific antibody formation.

Heavy and light chains for the IL1RAP×CD3 bispecific antibodies are shown below in Table 34.

TABLE 34

CDR sequences of 15 IL1RAP mAbs selected for

generation of IL1RAP × CD3 bispecific

antibodies (SEQ ID NOs in parentheses).

LC-
LC-
LC-

ID
HC-CDR1
HC-CDR2
HC-CDR3
CDR1
CDR2
CDR3

IAPB47
GYSFTSYW
IYPSDSYT
ARRNSAE
QSISND
YAS
QQSFT

(152)
(153)
NYADLDY
(155)
(156)
APLT

(154)

(157)

IAPB38
GFTFSNYA
INYGGGSK
AKDYGPF
QSVDDW
TAS
QQYHH

(158)
(159)
ALDY
(161)
(162)
WPLT

(160)

(163)

IAPB57
GGSISSST
IYFTGST
AKEDDSS
QGISSY
AAS
QQVNS

YY
(165)
GYYSFDY
(167)
(168)
YPLT

(164)

(166)

(169)

IAPB61
GVSISSST
IYFTGNT
GSLFGDY
QFISSN
GAS
QQYNN

YY
(171)
GYFDY
(173)
(174)
WPST

(170)

(172)

(175)

IAPB62
GYTFNTYA
INTNTGNP
ARRYFDW
QGISSW
AAS
QQANS

(176)
(177)
LLGAFDI
(179)
(168)
FPLT

(178)

(180)

IAPB3
GGTFSSYA
ISAIFGTA
ARGNSFH
QSVLYSS
WAS
QQYYS

(181)
(182)
ALWDYAF
NNKNY
(185)
TPLT

DY
(184)

(186)

(183)

IAPB17
GGTFSSYA
IIPIFGNA
ARTIIYL
QSVLYSS
WAS
QQYYS

(181)
(187)
DYVHILD
NNKNY
(185)
TPLT

Y
(184)

(186)

(188)

IAPB23
GFTFSNYW
IRYDGGSK
AKDAYPP
QSVSSY
DAS
QQRSN

(189)
(190)
YSFDY
(192)
(193)
WPLT

(191)

(194)

IAPB25
GFTFSSYA
ISGSGGST
AKGDEYY
QSISSY
AAS
QQSYS

(195)
(196)
YPDPLDY
(198)
(168)
TPLT

(197)

(199)

IAPB29
GFTFSNYA
ISGSGGST
AKEWSSY
QSISSY
AAS
QQSYS

(158)
(196)
FGLDY
(198)
(168)
TPLT

(200)

(199)

IAPB9
GGTFSSYA
ISPIFGTA
ARRYDNF
QSISSY
AAS
QQSYS

(181)
(201)
ARSGDLD
(198)
(168)
TPLT

Y

(199)

(202)

IAPB55
GVSISSST
IYFTGNT
GSLFGDY
QFISSN
GAS
QQYNN

YY
(171)
GYFDY
(173)
(174)
WPFT

(170)

(172)

(203)

IAPB63
GYTFNTYA
INTNTGNP
ARRYFDW
SSDVGDY
DVS
ASYAG

(176)
(177)
LLGAFDI
NY
(205)
NYNVV

(178)
(204)

(206)

IAPB64
GYTFNTYA
INTNTGNP
ARRYFDW
SSDVGDY
DVS
SSYAG

(176)
(177)
LLGAFDI
NY
(205)
NYNVV

(178)
(204)

(207)

IAPB65
GGTFSSYA
ISAIFGTA
ARHLHNA
QSVSNF
GAS
QQGKH

(181)
(182)
IHLDY
(209)
(174)
WPWT

(208)

(210)

VH and VL sequences of the 15 IL1RAP mAbs are shown below in Table 35.

TABLE 35

VH and VL sequences of IL1RAP mAbs for

generation of IL1RAP × CD3 bispecific

antibodies.

SEQ
VL Amino
SEQ

VH Amino
ID
Acid
ID

ID
Acid Sequence
NO:
Sequence
NO.

IAPB47
EVQLVQSGAEVKKPGESLK
211
EIVLTQSPGTLSL
212

ISCKGSGYSFTSYWIGWVR

SPGERATLSCRAS

QMPGKGLEWMGIIYPSDSY

QSISNDLNWYQQK

TRYSPSFQGQVTISADKSI

PGKAPKLLIYYAS

STAYLQWSSLKASDTAMYY

SLQSGVPSRFSGS

CARRNSAENYADLDYWGQG

GSGTDFTLTINSL

TLVTVSSASTKGPSVFPLA

QPEDFATYYCQQS

PCSRSTSESTAALGCLVKD

FTAPLTFGQGTKV

YFPEPVTVSWNSGALTSGV

EIKRTVAAPSVFI

HTFPAVLQSSGLYSLSSVV

FPPSDEQLKSGTA

TVPSSSLGTKTYTCNVDHK

SVVCLLNNFYPRE

PSNTKVDKRVESKYGPPCP

AKVQWKVDNALQS

PCPAPEAAGGPSVFLFPPK

GNSQESVTEQDSK

PKDTLMISRTPEVTCVVVD

DSTYSLSSTLTLS

VSQEDPEVQFNWYVDGVEV

KADYEKHKVYACE

HNATKPREEQFNSTYRVVK

VTHQGLSSPVTKS

SVLTVLHQDWLNGKEYKCK

FNRGEC

VSNKGLPSSIEKTISKAKG

QPREPQVYTLPPSQEEMTK

NQVSLTCLVKGFYPSDIAV

EWESNGQPENNYKTTPPVL

DSDGSFFLYSRLTVDKSRW

QEGNVFSCSVMHEALHNHY

TQKSLSLSLGK

IAPB38
EVQLLESGGGLVQPGGSLR
213
EIVLTQSPATLSL
214

LSCAASGFTFSNYAMNWV

SPGERATLSCRAS

RQAPGKGLEWVSGINYGG

QSVDDWLAWYQQK

GSKYYADSVKGRFTISRDN

PGQAPRLLIYTAS

SKNTLYLQMNSLRAEDTAV

NRATGIPARFSGS

YYCAKDYGPFALDYWGQG

GSGTDFTLTISSL

TLVTVSSASTKGPSVFPLA

EPEDFAVYYCQQY

PCSRSTSESTAALGCLVKD

HHWPLTFGQGTKV

YFPEPVTVSWNSGALTSGV

EIKRTVAAPSVFI

HTFPAVLQSSGLYSLSSVV

FPPSDEQLKSGTA

TVPSSSLGTKTYTCNVDHK

SVVCLLNNFYPRE

PSNTKVDKRVESKYGPPCP

AKVQWKVDNALQS

PCPAPEAAGGPSVFLFPPK

GNSQESVTEQDSK

PKDTLMISRTPEVTCVVVD

DSTYSLSSTLTLS

VSQEDPEVQFNWYVDGVEV

KADYEKHKVYACE

HNAKTKPREEQFNSTYRVV

VTHQGLSSPVTKS

SVLTVLHQDWLNGKEYKCK

FNRGEC

VSNKGLPSSIEKTISKAKG

QPREPQVYTLPPSQEEMTK

NQVSLTCLVKGFYPSDIAV

EWESNGQPENNYKTTPPVL

DSDGSFFLYSRLTVDKSRW

QEGNVFSCSVMHEALHNHY

TQKSLSLSLGK

IAPB57
QLQLQESGPGLVKPSETLS
215
DIQLTQSPSFLSA
216

LTCTVSGGSISSSTYYWGW

SVGDRVTITCRAS

IRQPPGKGLEWIGSIYFTG

QGISSYLAWYQQK

STDYNPSLKSRVSISVDTS

PGKAPKLLIYAAS

KNQFSLKLSSVTAADTAVY

TLQSGVPSRFSGS

YCAKEDDSSGYYSFDYWGQ

GSGTEFTLTISSL

GNLVTVSSASTKGPSVFPL

QPEDFATYYCQQV

APCSRSTSESTAALGCLVK

NSYPLTFGGGTKV

DYFPEPVTVSWNSGALTSG

EIKRTVAAPSVFI

VHTFPAVLQSSGLYSLSSV

FPPSDEQLKSGTA

VTVPSSSLGTKTYTCNVDH

SVVCLLNNFYPRE

KPSNTKVDKRVESKYGPPC

AKVQWKVDNALQS

PPCPAPEAAGGPSVFLFPP

GNSQESVTEQDSK

KPKDTLMISRTPEVTCVVV

DSTYSLSSTLTLS

DVSQEDPEVQFNWYVDGVE

KADYEKHKVYACE

VHNAKTKPREEQFNSTYRV

VTHQGLSSPVTKS

VSVLTVLHQDWLNGKEYKC

FNRGEC

KVSNKGLPSSIEKTISKAK

GQPREPQVYTLPPSQEEMT

KNQVSLTCLVKGFYPSDIA

VEWESNGQPENNYKTTPPV

LDSDGSFFLYSRLTVDKSR

WQEGNVFSCSVMHEALHNH

YTQKSLSLSLGK

IAPB61
QLQLQESGPGLVKPSETLS
217
EIVMTQSPATLSV
218

LTCTVSGVSISSSTYYWGW

PPGERATLSCRAS

LRQPPGMGLEWTGSIYFTG

QFISSNLAWYQQK

NTYYNPSLKSRVTISVDTS

PGQAPRLLIYGAS

RNQFSLKLSSVTAADTAVY

TRATGIPARFSGS

YCGSLFGDYGYFDYWGQGT

GSGTDFTLTISSL

LVTVSSASTKGPSVFPLAP

QSEDFAVYYCQQY

CSRSTSESTAALGCLVKDY

NNWPSTFGPGTKV

FPEPVTVSWNSGALTSGVH

DIKRTVAAPSVFI

TFPAVLQSSGLYSLSSVVT

FPPSDEQLKSGTA

VPSSSLGTKTYTCNVDHKP

SVVCLLNNFYPRE

SNTKVDKRVESKYGPPCPP

AKVQWKVDNALQS

CPAPEAAGGPSVFLFPPKP

GNSQESVTEQDSK

KDTLMISRTPEVTCVVVDV

DSTYSLSSTLTLS

SQEDPEVQFNWYVDGVEVH

KADYEKHKVYACE

NAKTKPREEQFNSTYRVVS

VTHQGLSSPVTKS

VLTVLHQDWLNGKEYKCKV

FNRGEC

SNKGLPSSIEKTISKAKGQ

PREPQVYTLPPSQEEMTKN

QVSLTCLVKGFYPSDIAVE

VVESNGQPENNYKTTPPVL

DSDGSFFLYSRLTVDKSRW

QEGNVFSCSVMHEALHNHY

TQKSLSLSLGK

IAPB62
QVQLVQSGSELKKPGASVK
219
DIQMTQSPSSVSA
220

VSCKASGYTFNTYAMNWV

SVGDRVTITCRAS

RQAPGQGLEWMGWINTNT

QGISSWLAWYQQK

GNPTYAQGFTGRFVFSLDT

PGKAPKLLIYAAS

SVSTAYLQISSLKAEDTAV

SLQSGVPSRFSGS

YYCARRYFDWLLGAFDIWG

GSGTDFTLTISSL

QGTMVTVSSASTKGPSVFP

QPEDFATYYCQQA

LAPCSRSTSESTAALGCLV

NSFPLTFGGGTKV

KDYFPEPVTVSWNSGALTS

EIKRTVAAPSVFI

GVHTFPAVLQSSGLYSLSS

FPPSDEQLKSGTA

VVTVPSSSLGTKTYTCNVD

SVVCLLNNFYPRE

HKPSNTKVDKRVESKYGPP

AKVQWKVDNALQS

CPPCPAPEAAGGPSVFLFP

GNSQESVTEQDSK

PKPKDTLMISRTPEVTCVV

DSTYSLSSTLTLS

VDVSQEDPEVQFNWYVDGV

KADYEKHKVYACE

EVHNAKTKPREEQFNSTYR

VTHQGLSSPVTKS

VVSVLTVLHQDWLNGKEYK

FNRGEC

CKVSNKGLPSSIEKTISKA

KGQPREPQVYTLPPSQEEM

TKNQVSLTCLVKGFYPSDI

AVEWESNGQPENNYKTTPP

VLDSDGSFFLYSRLTVDKS

RWQEGNVFSCSVMHEALHN

HYTQKSLSLSLGK

IAPB3
QVQLVQSGAEVKKPGSSVK
221
DIVMTQSPDSLAV
222

VSCKASGGTFSSYAISWVR

SLGERATINCKSS

QAPGQGLEWMGGISAIFGT

QSVLYSSNNKNYL

ANYAQKFQGRVTITADEST

AWYQQKPGQPPKL

STAYMELSSLRSEDTAVYY

LIYWASTRESGVP

CARGNSFHALWDYAFDYW

DRFSGSGSGTDFT

GQGTLVTVSSASTKGPSVF

LTISSLQAEDVAV

PLAPCSRSTSESTAALGCL

YYCQQYYSTPLTF

VKDYFPEPVTVSWNSGALT

GQGTKVEIKRTVA

SGVHTFPAVLQSSGLYSLS

APSVFIFPPSDEQ

SVVTVPSSSLGTKTYTCNV

LKSGTASVVCLLN

DHKPSNTKVDKRVESKYGP

NFYPREAKVQWKV

PCPPCPAPEAAGGPSVFLF

DNALQSGNSQESV

PPKPKDTLMISRTPEVTCV

TEQDSKDSTYSLS

VVDVSQEDPEVQFNWYVDG

STLTLSKADYEKH

VEVHNAKTKPREEQFNSTY

KVYACEVTHQGLS

RVVSVLTVLHQDWLNGKEY

SPVTKSFNRGEC

KCKVSNKGLPSSIEKTISK

AKGQPREPQVYTLPPSQEE

MTKNQVSLTCLVKGFYPSD

IAVEWESNGQPENNYKTTP

PVLDSDGSFFLYSRLTVDK

SRWQEGNVFSCSVMHEALH

NHYTQKSLSLSLGK

IAPB17
QVQLVQSGAEVKKPGSSVK
223
DIVMTQSPDSLAV
222

VSCKASGGTFSSYAISWVR

SLGERATINCKSS

QAPGQGLEWMGGIIPIFGN

QSVLYSSNNKNYL

ANYAQKFQGRVTITADEST

AWYQQKPGQPPKL

STAYMELSSLRSEDTAVYY

LIYWASTRESGVP

CARTIIYLDYVHILDYWGQ

DRFSGSGSGTDFT

GTLVTVSSASTKGPSVFPL

LTISSLQAEDVAV

APCSRSTSESTAALGCLVK

YYCQQYYSTPLTF

DYFPEPVTVSWNSGALTSG

GQGTKVEIKRTVA

VHTFPAVLQSSGLYSLSSV

APSVFIFPPSDEQ

VTVPSSSLGTKTYTCNVDH

LKSGTASVVCLLN

KPSNTKVDKRVESKYGPPC

NFYPREAKVQWKV

PPCPAPEAAGGPSVFLFPP

DNALQSGNSQESV

KPKDTLMISRTPEVTCVVV

TEQDSKDSTYSLS

DVSQEDPEVQFNWYVDGVE

STLTLSKADYEKH

VHNAKTKPREEQFNSTYRV

KVYACEVTHQGLS

VSVLTVLHQDWLNGKEYKC

SPVTKSFNRGEC

KVSNKGLPSSIEKTISKAK

GQPREPQVYTLPPSQEEMT

KNQVSLTCLVKGFYPSDIA

VEWESNGQPENNYKTTPPV

LDSDGSFFLYSRLTVDKSR

WQEGNVFSCSVMHEALHNH

YTQKSLSLSLGK

IAPB23
EVQLLESGGGLVQPGGSLR
224
EIVLTQSPATLSL
225

LSCAASGFTFSNYWMNWV

SPGERATLSCRAS

RQAPGKGLEWVSAIRYDGG

QSVSSYLAWYQQK

SKYYADSVKGRFTISRDNS

PGQAPRLLIYDAS

KNTLYLQMNSLRAEDTAV

NRATGIPARFSGS

YYCAKDAYPPYSFDYWGQ

GSGTDFTLTISSL

GTLVTVSSASTKGPSVFPL

EPEDFAVYYCQQR

APCSRSTSESTAALGCLVK

SNWPLTFGQGTKV

DYFPEPVTVSWNSGALTSG

EIKRTVAAPSVFI

VHTFPAVLQSSGLYSLSSV

FPPSDEQLKSGTA

VTVPSSSLGTKTYTCNVDH

SVVCLLNNFYPRE

KPSNTKVDKRVESKYGPPC

AKVQWKVDNALQS

PPCPAPEAAGGPSVFLFPP

GNSQESVTEQDSK

KPKDTLMISRTPEVTCVVV

DSTYSLSSTLTLS

DVSQEDPEVQFNWYVDGVE

KADYEKHKVYACE

VHNAKTKPREEQFNSTYRV

VTHQGLSSPVTKS

VSVLTVLHQDWLNGKEYKC

FNRGEC

KVSNKGLPSSIEKTISKAK

GQPREPQVYTLPPSQEEMT

KNQVSLTCLVKGFYPSDIA

VEWESNGQPENNYKTTPPV

LDSDGSFFLYSRLTVDKSR

WQEGNVFSCSVMHEALHNH

YTQKSLSLSLGK

IAPB25
EVQLLESGGGLVQPGGSLR
226
DIQMTQSPSSLSA
227

LSCAASGFTFSSYAMSWVR

SVGDRVTITCRAS

QAPGKGLEWVSAISGSGGS

QSISSYLNWYQQK

TYYADSVKGRFTISRDNSK

PGKAPKLLIYAAS

NTLYLQMNSLRAEDTAVY

SLQSGVPSRFSGS

YCAKGDEYYYPDPLDYWG

GSGTDFTLTISSL

QGTLVTVSSASTKGPSVFP

QPEDFATYYCQQS

LAPCSRSTSESTAALGCLV

YSTPLTFGQGTKV

KDYFPEPVTVSWNSGALT

EIKRTVAAPSVFI

SGVHTFPAVLQSSGLYSLS

FPPSDEQLKSGTA

SVVTVPSSSLGTKTYTCN

SVVCLLNNFYPRE

VDHKPSNTKVDKRVESKYG

AKVQWKVDNALQS

PPCPPCPAPEAAGGPSVFL

GNSQESVTEQDSK

FPPKPKDTLMISRTPEVTC

DSTYSLSSTLTLS

VVVDVSQEDPEVQFNWYVD

KADYEKHKVYACE

GVEVHNAKTKPREEQFNST

VTHQGLSSPVTKS

YRVYSVLTVLHQDWLNGKE

FNRGEC

YKCKVSNKGLPSSIEKTIS

KAKGQPREPQVYTLPPSQE

EMTKNQVSLTCLVKGFYPS

DIAVEWESNGQPENNYKTT

PPVLDSDGSFFLYSRLTVD

KSRWQEGNVFSCSVMHEAL

HNHYTQKSLSLSLGK

IAPB29
EVQLLESGGGLVQPGGSLR
228
DIQMTQSPSSLSA
227

LSCAASGFTFSNYAMSWVR

SVGDRVTITCRAS

QAPGKGLEWVSAISGSGGS

QSISSYLNWYQQK

TYYADSVKGRFTISRDNSK

PGKAPKLLIYAAS

NTLYLQMNSLRAEDTAVY

SLQSGVPSRFSGS

YCAKEWSSYFGLDYWGQG

GSGTDFTLTISSL

TLVTVSSASTKGPSVFPLA

QPEDFATYYCQQS

PCSRSTSESTAALGCLVKD

YSTPLTFGQGTKV

YFPEPVTVSWNSGALTSGV

EIKRTVAAPSVFI

HTFPAVLQSSGLYSLSSVV

FPPSDEQLKSGTA

TVPSSSLGTKTYTCNVDHK

SVVCLLNNFYPRE

PSNTKVDKRVESKYGPPCP

AKVQWKVDNALQS

PCPAPEAAGGPSVFLFPPK

GNSQESVTEQDSK

PKDTLMISRTPEVTCVVVD

DSTYSLSSTLTLS

VSQEDPEVQFNWYVDGVEV

KADYEKHKVYACE

HNAKTKPREEQFNSTYRVV

VTHQGLSSPVTKS

SVLTVLHQDWLNGKEYKCK

FNRGEC

VSNKGLPSSIEKTISKAKG

QPREPQVYTLPPSQEEMTK

NQVSLTCLVKGFYPSDIAV

EWESNGQPENNYKTTPPVL

DSDGSFFLYSRLTVDKSRW

QEGNVFSCSVMHEALHNHY

TQKSLSLSLGK

IAPB9
QVQLVQSGAEVKKPGSSVK
229
DIQMTQSPSSLSA
227

VSCKASGGTFSSYAISWVR

SVGDRVTITCRAS

QAPGQGLEWMGWISPIFGT

QSISSYLNWYQQK

ANYAQKFQGRVTITADEST

PGKAPKLLIYAAS

STAYMELSSLRSEDTAVYY

SLQSGVPSRFSGS

CARRYDNFARSGDLDYWG

GSGTDFTLTISSL

QGTLVTVSSASTKGPSVFP

QPEDFATYYCQQS

LAPCSRSTSESTAALGCLV

YSTPLTFGQGTKV

KDYFPEPYTVSWNSGALTS

EIKRTVAAPSVFI

GVHTFPAVLQSSGLYSLSS

FPPSDEQLKSGTA

VVTVPSSSLGTKTYTCNVD

SVVCLLNNFYPRE

HKPSNTKVDKRVESKYGPP

AKVQWKVDNALQS

CPPCPAPEAAGGPSVFLFP

GNSQESVTEQDSK

PKPKDTLMISRTPEVTCVV

DSTYSLSSTLTLS

VDVSQEDPEVQFNWYVDGV

KADYEKHKVYACE

EVHNAKTKPREEQFNSTYR

VTHQGLSSPVTKS

VVSVLTVLHQDWLNGKEYK

FNRGEC

CKVSNKGLPSSIEKTISKA

KGQPREPQVYTLPPSQEEM

TKNQVSLTCLVKGFYPSDI

AVEWESNGQPENNYKTTPP

VLDSDGSFFLYSRLTVDKS

RWQEGNVFSCSVMHEALHN

HYTQKSLSLSLGK

IAPB55
QLQLQESGPGLVKPSETLS
633
EIVMTQSPATLSV
230

LTCTVSGVSISSSTYYWGW

SPGERATLSCRAS

LRQPPGMGLEWTGSIYFTG

QFISSNLAWYQQK

NTYYNPSLKSRVTISVDTS

PGQAPRLLIYGAS

RNQFSLKLSSVTAADTAVY

TRATGIPARFSGS

YCGSLFGDYGYFDYWGQGT

GSGTDFTLTISSL

LVTVSSASTKGPSVFPLAP

QSEDFAVYYCQQY

CSRSTSESTAALGCLVKDY

NNWPFTFGPGHCV

FPEPVTVSWNSGALTSGVH

DIKRTVAAPSVFI

TFPAVLQSSGLYSLSSVVT

FPPSDEQLKSGTA

VPSSSLGTKTYTCNVDHKP

SVVCLLNNFYPRE

SNTKVDKRVESKYGPPCPP

AKVQWKVDNALQS

CPAPEAAGGPSVFLEPPKP

GNSQESVTEQDSK

KDTLMISRTPEVTCVVVDV

DSTYSLSSTLTLS

SQEDPEVQFNWYVDGVEVH

KADYEKHKVYACE

NAKTKPREEQFNSTYRVVS

VTHQGLSSPVTKS

VLTVLHQDWLNGKEYKCKV

FNRGEC

SNKGLPSSIEKTISKAKGQ

PREPQVYTLPPSQEEMTKN

QVSLTCLVKGFYPSDIAVE

WESNGQPENNYKTTPPVLD

SDGSFFLYSRLTVDKSRWQ

EGNVFSCSVMHEALHNHYT

QKSLSLSLGK

IAPB63
QVQLVQSGSELKKPGASVK
219
QSALTQPRSVSGS
231

VSCKASGYTFNTYAMNWV

PGHSVTISCTGTS

RQAPGQGLEWMGWINTNT

SDVGDYNYVSWYQ

GNPTYAQGFTGRFVFSLDT

QRPGKVPKLLIYD

SVSTAYLQISSLKAEDTAV

VSKRPSGVPDRFS

YYCARRYFDWLLGAFDIWG

GSKSGNTASLTIS

QGTMVTVSSASTKGPSVFP

GLQAEDEAIYFCA

LAPCSRSTSESTAALGCLV

SYAGNYNVVFGGG

KDYFPEPVTVSWNSGALTS

TKLTVLGQPKAAP

GVHTFPAVLQSSGLYSLSS

SVTLFPPSSEELQ

VVTVPSSSLGTKTYTCNVD

ANKATLVCLISDF

HKPSNTKVDKRVESKYGPP

YPGAVTVAWKADS

CPPCPAPEAAGGPSVFLFP

SPVKAGVETTTPS

PKPKDTLMISRTPEVTCVV

KQSNNKYAASSYL

VDVSQEDPEVQFNWYVDGV

SLTPEQWKSHRSY

EVHNAKTKPREEQFNSTYR

SCQVTHEGSTVEK

VVSVLTVLHQDWLNGKEYK

TVAPTECS

CKVSNKGLPSSIEKTISKA

KGQPREPQVYTLPPSQEEM

TKNQVSLTCLVKGFYPSDI

AVEWESNGQPENNYKTTPP

VLDSDGSFFLYSRLTVDKS

RWQEGNVFSCSVMHEALHN

HYTQKSLSLSLGK

IAPB64
QVQLVQSGSELKKPGASVK
219
QSALTQPRSVSGS
232

VSCKASGYTFNTYAMNWV

PGHSVTISCTGTS

RQAPGQGLEWMGWINTNT

SDVGDYNYVSWYQ

GNPTYAQGFTGRFVFSLDT

QRPGKVPKLLIYD

SVSTAYLQISSLKAEDTAV

VSKRPSGVPDRFS

YYCARRYFDWLLGAFDIWG

GSKSGNTASLTIS

QGTMVTVSSASTKGPSVFP

GLQAEDEAIYFCS

LAPCSRSTSESTAALGCLV

SYAGNYNVVFGGG

KDYFPEPVTVSWNSGALTS

TKLTVLGQPKAAP

GVHTFPAVLQSSGLYSLSS

SVTLFPPSSEELQ

VVTVPSSSLGTKTYTCNVD

ANKATLVCLISDF

HKPSNTKVDKRVESKYGPP

YPGAVTVAWKADS

CPPCPAPEAAGGPSVFLFP

SPVKAGVETTTPS

PKPKDTLMISRTPEVTCVV

KQSNNKYAASSYL

VDVSQEDPEVQFNWYVDGV

SLTPEQWKSHRSY

EVHNAKTKPREEQFNSTYR

SCQVTHEGSTVEK

VVSVLTVLHQDWLNGKEYK

TVAPTECS

CKVSNKGLPSSIEKTISKA

KGQPREPQVYTLPPSQEEM

TKNQVSLTCLVKGFYPSDI

AVEWESNGQPENNYKTTPP

VLDSDGSFFLYSRLTVDKS

RWQEGNVFSCSVMHEALH

NHYTQKSLSLSLGK

IAPB65
QVQLVQSGAEVKKPGSSVK
233
EIVLTQSPATLSL
234

VSCKASGGTFSSYAISWVR

SPGERATLSCRAS

QAPGQGLEWMGGISAIFGT

QSVSNFLAWYQQK

ANYAQKFQGRVTITADEST

PGQAPRLLIYGAS

STAYMELSSLRSEDTAVYY

NRATGIPARFSGS

CARHLHNAIHLDYWGQGT

GSGTDFTLTISSL

LVTVSSASTKGPSVFPLAP

EPEDFAVYYCQQG

CSRSTSESTAALGCLVKDY

KHWPWTFGQGTKV

FPEPVTVSWNSGALTSGVH

EIKRTVAAPSVFI

TFPAVLQSSGLYSLSSVVT

FPPSDEQLKSGTA

VPSSSLGTKTYTCNVDHKP

SVVCLLNNFYPRE

SNTKVDKRVESKYGPPCPP

AKVQWKVDNALQS

CPAPEAAGGPSVFLFPPKP

GNSQESVTEQDSK

KDTLMISRTPEVTCVVVDV

DSTYSLSSTLTLS

SQEDPEVQFNWYVDGVEVH

KADYEKHKVYACE

NAKTKPREEQFNSTYRVVS

VTHQGLSSPVTKS

VLTVLHQDWLNGKEYKCKV

FNRGEC

SNKGLPSSIEKTISKAKGQ

PREPQVYTLPPSQEEMTKN

QVSLTCLVKGFYPSDLAVE

WESNGQPENNYKTTPPVLD

SDGSFFLYSRLTVDKSRWQ

EGNVFSCSVMHEALHNHYT

QKSLSLSLGK

4-1 Evaluation of IL1RAP Bispecific Antibodies in a Whole Blood T Cell Cytotoxicity Assay

Methods: Tumor cell line LAMA-84 was washed with DPBS before incubation with CFSE (resuspended in 150 μL DMSO and diluted 1:10,000) at 10×10⁶cells/mL of CFSE for 8 minutes at room temperature. Staining was quenched with HI FBS and washed in media before resuspension in complete media at 2×10⁵cells/mL. Equal volumes of whole blood and CFSE-stained tumor cells were combined in each well of a 96-well plate. After the addition of antibody (resuspended in 10 μL of DPBS) in respective wells, all plates were incubated at 37° C. with 5% CO₂for 48 hours. Undiluted CD25-PE was added directly to each well of whole blood and incubated for 30 minutes at room temperature, protected from light. Cells were then centrifuged at 1500 rpm for 5 minutes and lysed with 200 uL of multi-specie red lysis buffer, repeated 4 more times. Cells were washed in DPBS once and stained with Live/Dead Near-IR (diluted in DPBS) for 15 minutes (Table 36). Each well was washed and resuspended in FACS buffer before the cells were acquired on a BD FACS CANTO II.

TABLE 36

Whole Blood T cell cytotoxicity staining panel

Antigen
Channel
Clone
Dilution
Source
Catalog #

CD25
PE
BC96
1:100
Biolegend
302606

CD4
PerCP
RPA-T4
1:100
Biolegend
300528

CD8
APC
HIT8a
1:100
Biolegend
300912

Live/Dead
APC-Cy7
NA*
1:200
Invitrogen
L34976

*NA = not applicable

The cell acquisition on the FACS CANTO II (Becton, Dickinson and Company, Franklin Lakes, NJ) was limited to 1×10⁴tumor cells. Tumor cell death was assessed by gating on FSC and SSC to identify cell populations, then CFSE positive tumor events, and finally Live/Dead Near-IR to assess tumor cytotoxicity. T-cell activation was assessed by gating on FSC and SSC to identify cell populations, CFSE negative events, Near-IR negative events to assess live cells, then CD25 positive cells. The percentage of either dead tumor cells or CD25 positive cells were graphed using GraphPad Prism 6 and analyzed with a non-linear curve fit. Additional statistical analysis was evaluated in the program “R” to determine population EC₅₀values using a nonlinear mixed-effects model.

Results: To mimic physiological peripheral whole blood conditions containing sIL1RAP, IC3B19 (IAPB57×CD3B219) and IC3B34 (IAPB57×CD3B376) were evaluated in an ex vivo T cell redirection assay utilizing whole blood. Exogenously added IL1RAP⁺ LAMA-84 cell line (2×10⁴cells/well) was added to healthy control human peripheral whole blood (n=15 donor samples) with IC3B19, IC3B34, and null arm control bispecific antibodies for 48 hours. As shown in FIG. 52, IC3B19 and IC3B34 induced similar T-cell mediated cytotoxicity of IL1RAP⁺ LAMA-84 cell line ex vivo after 48 hours. The EC₅₀values for IC3B19 and IC3B34 cytotoxicity, represented FIG. 52 were 2.097 and 2.762, respectively (Table 37). Similarly, the EC₅₀values for T-cell activation (CD25) by IC3B19 and IC3B34 represented by FIG. 53 were 4.237 and 7.825 nM, respectively (Table 38). All of the null arm control bispecific antibodies did not induce detectable levels of cytotoxicity or T-cell activation as shown in FIG. 52 and FIG. 53. The general level of cytotoxicity and T-cell activation were similar between both IC3B19 and IC3B34.

TABLE 37

IC3B19 and IC3B34 Cytotoxicity EC50 (nM)

Concentrations in the LAMA-84 cell line

Effective Concentration (nM)
IC3B19
IC3B34

EC₅₀
2.097
2.762

TABLE 38

IC3B19 and IC3B34 T-cell Activation EC50 (nM)

Concentrations in the LAMA-84 cell line

Effective Concentration (nM)
IC3B19
IC3B34

EC50
4.237
7.825

4-2 Evaluation of Cytokine Release by IL1RAP×CD3 Bispecific Abs in a Whole Blood T Cell Cytotoxicity Assay at 24 and 48 Hours

Methods: Frozen supernatants from 24 hour and 48 hour samples from Example 4-1 were thawed on wet ice. Prior to use, plates were centrifuged at 500 g for 5 minutes at 4° C., then placed back on wet ice. Serial dilutions were prepared in U-bottom 96-well plates (Falcon), using MSD Diluent 2 at 1:2, 1:25, and 1:1000 dilution ratios for ELISA.

While supernatants were being centrifuged, MSD Assay Plates (ProInflammatory Panel I V-Plex, Catalog #K15049G-4, Lot #K008572) were pre-washed per manufacturer's protocol. A standard curve was prepared by reconstituting the provided calibrator in 1.0 mL of MSD Diluent 2. Fifty microliters of each sample or standard was added directly to pre-washed MSD plates. Subsequent incubations and washes were all carried out per manufacturer's protocol. Assay plates were read on the MSD Sector Imager.

Two methods were used to compare response between IC3B19 and IC3B34:

Linear Regression with Mixed Effects

In this method, a linear regression model with fixed effects for compound, dose, and compound by dose interaction and random effects that take into account the paired donors was constructed. Post-hoc comparisons were then between IC3B19 and IC3B34 were made at each concentration and p-values were appropriately adjusted for multiple comparisons.

Non-Linear (Four-Parameter) Regression with Mixed Effects

IC3B19 and IC3B34 were modeled together using a non-linear regression model, with fixed effect parameters for minimum, maximum, log EC50, and slope for each compound, and a random effect term that takes into account paired donor effect. Point estimates and their confidence intervals were extracted and plotted for visual comparison from these separate models. Testing significant differences for each parameter was done by constructing a model that used a joint estimate for the parameter of interest, then comparing that model to the original model using a log-likelihood test. To elaborate, there is a model with parameters for IC3B19 minimum, IC3B19 maximum, IC3B19 log EC50, IC3B19 slope, IC3B34 minimum, IC3B34 maximum, IC3B34 log EC50, and IC3B34 slope. To check whether the minimums are the significantly different, a second model is constructed that has all the same parameter except that there is a single shared parameter for the minimum. The log likelihood (an overall measure of how well the model fits the data) of the first model is compared to the log likelihood of the second model. If they are not significantly different from each other, then we can conclude that the minimums are not significantly different from each other. Similarly, if the log likelihoods are significantly different from one another, then we would conclude that the minimums are significantly different.

In a number of cases, models or parameter comparisons were inestimable for one of two reasons: 1. the data was to intractable to calculate the four-parameter logistic regression itself, or 2. the difference between the parameter estimates was so large that is caused the joint model to fail. In the first case, results are not reported and in the summary table they are indicated with “NA”. In the second, results are also not reported, but they are indicated in the summary table as “Sig.”, as short-hand for significant. As the 95% confidence intervals around these parameters are individually estimable and do not overlap, we can consider them significantly different but the exact p-value is unknown.

Differences are considered statistically significant at p-value less than or equal to 0.05. All analyses were conducted in R, version 3.3.2.

Results: IC3B19 and IC3B34 mediated engagement of T cells and IL1RAP+ target cell line LAMA-84 (endogenous and exogenously added tumor cells) resulted in T cell cytokine release. To determine cytokine release difference between IC3B19 and IC3B34, supernatants were evaluated for 10 pro-inflammatory cytokines from whole blood (n=15 donors) cytotoxicity and T-cell activation assays with exogenous LAMA-84 IL1RAP+ tumor cell line added, as discussed in section example 1. Of the 10 cytokines evaluated, most cytokines resulted in a measurable dose dependent release in response to both IC3B19 and IC3B34, except for IL-8 and IL-12p70. As shown in Table 39, IL-10, IL-2, TNF-α, and INF-γ resulted in the most potent EC50 (nM) release. In addition, the majority of cytokines and at both time points, the IC3B34 molecule resulted in a less potent EC50 (nM) that was statistically significant. The exception were, IL-4 (24 hours), IL-6 (24 and 48 hours), IL-8 (24 and 48 hours), IL-12p70 (24 and 48 hours), IL-13 (24 hours), INF-γ (24 and 48 hours), and TNF-α (24 hours), which did not have a statistically significant difference in EC50 (nM) values between IC3B19 and IC3B34.

TABLE 39

IC3B19 and IC3B34 Four Parameter Logistic Regression Summary Table.

Bold text denotes statistical significant difference in EC50 (nM)

values between IC3B19 and IC3B34. Results of individual cytokine

release experiments are shown in the FIG. Nos. indicated.

Cytokine

FIG. No.

Experiment
EC50
Slope
Minimum
Maximum

IL-1beta

FIG. 36

24 hours

0.0027

0.3046

0.0268

0.2033

IL-1beta

FIG. 37

48 hours

0.0010

0.3951
0.2543
0.0528

IL-2

FIG. 38

24 hours

0.0001

0.0256

0.3103
0.6466

IL-2

FIG. 39

48 hours

Sig.

0.2079
0.0158

0.0008

IL-4

FIG. 40

24 hours
0.2415
0.4668
0.2942

0.0043

IL-4

FIG. 41

48 hours
NA
NA
NA
NA

IL-6

FIG. 42

24 hours

Sig.

0.0819
0.1151
0.5224

IL-6

FIG. 43

48 hours
NA
NA
NA
NA

IL-8

FIG. 44

24 hours
NA
NA
NA
NA

IL-8

FIG. 45

48 hours
NA
NA
NA
NA

IL-10

FIG. 46

24 hours

<0.0001

0.0010

0.3406
0.7036

IL-10

FIG. 47

48 hours

<0.0001
0.2702

0.0011

0.0004

IL-12p70

FIG. 48

24 hours
NA
NA
NA
NA

IL-12p70

FIG. 49

48 hours
NA
NA
NA
NA

IL-13

FIG. 50

24 hours
NA
NA
NA
NA

IL-13

FIG. 51

48 hours

0.0106

0.8268

<0.0001

<0.0001

IFN-gamma

FIG. 52

24 hours

0.0065

0.6920
0.4644
0.3179

IFN-gamma

FIG. 53

48 hours

0.0266

0.1678
0.2695

0.0319

TNF-alpha

FIG. 54

24 hours

0.0016

0.0128

0.1443
0.7895

TNF-alpha

FIG. 55

48 hours

0.0004

0.5553

0.0043

0.5216

4-3 Evaluation of IL1RAP Bispecific Antibodies in a Caspase Cytotoxicity Assay

T-cell mediated killing of IC3B19 and IC3B34 was measured using a second type of cell toxicity assay. The caspase cytotoxicity assay indirectly measures cell killing via cleavage of a fluorescent substrate by active caspase 3/7. Cleavage of the substrate results in a fluorescent DNA dye, with fluorescence restricted to the cell nucleus. Repeated fluorescence measurements are taken in each well throughout the course of the assay, using a motorized 10× objective, capable of precisely imaging well(s) at the same coordinates. Target cell populations are identified based on defined size restrictions.

Frozen Pan CD3+ T-cells (purchased from Biological Specialty Corporation, Colmar, PA) were isolated by negative selection from normal healthy donors. Target cells (NCI-H1975 cells, a human lung adenocarcinoma cell line expressing IL1RAP), were cultured in RPMI 1640/Glutamax (25 mM HEPES buffer) medium supplemented with 10% heat inactivated FBS, 1 mM sodium pyruvate, and 0.1 mM non-essential amino acids (Life Technologies).

Target cells were plated at 20,000 cells/well in 50 ul of phenol-red free RPMI media containing supplements 16 hours prior to experiment initiation into tissue culture-treated, clear flat bottom plates (Costar). The cells were allowed to incubate at room temperature (RT) for 20 min to allow for even distribution of cells in the wells, and then the cells were allowed to incubate at 37° C. and 5% CO2 overnight.

On the day of the experiment, T-cells were counted and diluted to 1.0*10{circumflex over ( )}6 cells/ml in phenol red free RPMI media containing supplements and combined with Nuc-View TM488 Caspase-3/7 Substrate (Essen Biosciences) for a working concentration of 10 uM. 100 ul of the combined T-cells and the Caspase-3/7 substrate were added to each well for a final concentration of 5 uM of the Caspase-3/7 substrate.

IC3B19 (IAPB57×CD3B219), IC3B34 (IAPB57×CD3B376), CD3B288 (Null×CD3B219), and IAPB57×Null (IAPB101) bispecific antibodies were prepared at 4× final concentration in phenol red free RPMI media containing supplements and 50 uL of the antibodies were added to each well. After a 20 min incubation at RT to allow for even distribution in the wells, plates were transferred to the Zoom Incucyte instrument (Essen Bioscience). The Incucyte Instrument resides in a humidified incubator set at 37° C., 5% CO2.

A processing definition on the Incucyte was designed for the NCI-H1975 cells. This processing definition clearly identifies caspase activity of target cells, while excluding the T-cells by size. Measurements were taken at TO and every six hours for up to 120 hours. The maximum signal was determined to be at 72 hours post treatment at which time the data was analyzed.

After the assay was complete, each plate was analyzed using the NCI-H1975 processing definition. Raw fluorescent data was exported from the Incucyte Zoom software, and pasted into GraphPad Prism (GraphPad Software, Inc., La Jolla, CA). Caspase 3/7 activity was determined by calculating the area under the curve (AUC) for each well in GraphPad. AUC values were plotted as a function of Log 10 nM antibody concentrations. The EC50 for each concentration response curve, in nanomolar (nM), was reported following non-linear regression (Sigmoidal dose-response-variable slope). Each assay contained two technical replicates and four measurements within each technical replicate.

Both IC3B19 and IC3B34, but not the bispecific antibodies with null arms (IAPB57×B23B49 or B23B39×CD3B219), induced target specific cytoxicity in NCI-H1975 cells (FIG. 56). In this assay, the cytotoxicity EC50s vary three-fold between IC3B19 and IC3B34, with values of 0.018 and 0.057 nM, respectively.

4-4 Efficacy of IAPB57×CD3B376 in 111975 Human NSCLC Xenografts in T Cell Humanized NSG Mice

Efficacy of IAPB57×CD3B376 bispecific antibody IC3B34 was evaluated in established H1975 human Non-small cell lung cancer (NSCLC) xenografts in female NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice humanized with human T cells. IAPB57×CD3B376 at 0.1, 0.35 or 1 mg/kg or Null×CD3 antibody control was dosed on Days 14, 18, 21 and 25 for a total of 4 doses. On day 29 post-tumor implant, which was the last date of the study when ten animals remained per group, tumor growth inhibition (% TGI) was calculated. Statistically significant tumor growth inhibition was observed with IAPB57×CD3B376 at 0.35 and 1 mg/kg with 63% and 89% TGI, respectively compared to Null×CD3 control (Two-way ANOVA with Bonferroni test, p<0.0001, FIG. 57).

5 CD33 Antibodies
5-1 Antigen Generation

The human and cyno CD33 proteins were produced with or without a mutated monomeric form of human serum albumin (HSA), Uniprot P02768 with a C58S mutation, fused at the C-terminus for immunizations and assays. The cDNAs encoding the CD33 protein antigens with a six-histidine tag (SEQ ID NO: 596) were synthetically synthesized and cloned into a mammalian secretion expression vector under the Actin promoter using standard molecular biology techniques.

The full-length human CD33 extracellular domain (ECD) derived from Uniprot P20138 (SEQ ID NO:235) (human CD33 ECD) was fused at the N-terminus with a signal sequence and with or without the HSA, followed by a six histidine tag (SEQ ID NO: 596) at the C-terminus, (hCD33 ECD with HSA and hCD33 ECD only). The human CD33 ECD expression construct was transiently transfected into HEK293 derived cells, Expi293 (Gibco/Thermo Fisher Scientific; Waltham, MA) using Expifectamine according to manufacturer protocol. Cells were incubated 5 days at 37° C. with 8% CO2 on an orbital shaker before harvesting. The expressed cells were removed by centrifugation and the soluble CD33 was purified from the media using immobilized metal affinity chromatography using Ni Sepharose 6 Fast Flow resin (GE Healthcare; Little Chalfont, United Kingdom) followed by Superdex 200 preparative size exclusion chromatography (SEC) (GE Healthcare) in Dubelcco's Phosphate Saline buffer pH 7.2 (1×DPBS). SEC elution fractions excluding any disulfide aggregates were combined and sterile filtered to yield the final protein for immunization and CD33 assays. Protein concentration was determined by A280 and quality of purified protein was assessed by SDS-PAGE and analytical SEC (Phenomenex; Torrance, CA). Endotoxin measurements were performed using EndoSafe-PTS Cartridges, a chromogenic LAL assay (Charles River; Wilmington, MA).

The human CD33 ECD subdomain proteins, hCD33 V-domain-HSA, hCD33 V-domain-his, hCD33 C2 domain-HSA, and hCD33 C2 domain-His, were similarly constructed, expressed and purified as the full-length human CD33 ECD.

Cyno CD33 constructs for immunization and cross selectivity assays, cyno CD33 ECD-HSA, cyno CD33-His, were also generated based on the Genbank sequence XP_0055901381 Cyno CD33 protein expression and purification were same as the human CD33 proteins.

The CD33 antigens for screening were biotinylated in 50 mM Na Phosphate pH 7.2 using SureLink Chromagenic Biotin Labeling kit (SeraCare KPL) according to manufacturer conditions. Briefly, a biotin stock of 25 mM was added to the CD33 protein at a 4:1 molar ratio of biotin to protein and incubated at room temperature for 30 minutes with gentle rotation and then switched to 4° C. for 2 more hours. Unincorporated biotin was removed by buffer exchange into 1×DPBS. Protein concentration and biotin incorporation was determined by measuring at A280 nm and A354 nm using NanoDrop. See Table 40 for the sequences of each of the antigens described above.

TABLE 40

Antigen Sequences

Protein Name
Protein ID
SEQ ID NO

Cyno CD33 ECD-HSA
C33W1
236

Human CD33 ECD-HSA
C33W2
237

Human CD33-V-HSA
C33W3
238

Human CD33-C2-HSA
C33W4
239

Human CD33-V-His
C33W8
240

Human CD33 C2-His
C33W9
241

Human CD33 ECD-His
C33W49
242

Cyno CD33 ECD-His
C33W50
243

Human CD33 full length

244

Cyno CD33 full length

245

5-2 Generation of CD33 Expressing Isogenic Cell Lines

Human and cyno CD33 expressing cell lines were generated using lentivirus (Genecopoeia; Rockville, MD) containing full length human CD33 or cyno CD33 and puromycin for selection of CD33 positive cells. HEK293F cells (ATCC), negative for CD33, were transduced with lentiviral particles to overexpress human CD33 and cyno CD33. Following transduction, cells positively expressing CD33 and the resistance marker, were selected by treating pooled cells, grown in DMEM+10% HI FBS (Life Technologies; Carlsbad, CA) and supplemented with varying concentrations of Puromycin (Life Technologies).

In addition to the HEK generated cell lines, several commercial cell lines were used for binding and cellular toxicity assays. These included MOLM13, KG1, SH2, OCIAML3 and MV411 and were obtained from either American Type Culture Collection or Deutsche Sammlung von Mikrooranismen and Zellkulturen, and cultured at 37° C., 5% CO2 in complete RPMI culture media with 10% FBS.

5-3 Immunization Campaigns
OmniRat

A human immunoglobulin transgenic rat strain (OmniRat®; Ligand Pharmaceuticals; San Diego, CA) was used to develop human CD33 monoclonal antibody expressing hybridoma cells. The OmniRat® contains a chimeric human/rat IgH locus (comprising 22 human V_Hs, all human D and J_Hsegments in natural configuration linked to the rat CH locus) together with fully human IgL loci (12 Vκs linked to Jκ-Cκ and 16 Vλs linked to Jλ-Cλ) (see e.g., Osborn, et al. (2013) J Immunol 190(4): 1481-1490). Accordingly, the rats exhibit reduced expression of rat immunoglobulin, and in response to immunization, the introduced human heavy and light chain transgenes undergo class switching and somatic mutation to generate high affinity chimeric human/rat IgG monoclonal antibodies with fully human variable regions. The preparation and use of OmniRat®, and the genomic modifications carried by such rats, is described in PCT Publication WO 2014/093908 to Bruggemann et al.

When immunized with recombinant human and cynomolgus CD33 (huCD33 ECD-HSA and cyno CD33 ECD-HSA respectively), this transgenic rat produces chimeric human-rat IgG antibodies to human CD33, some of which also bind to cynomolgus CD33.

Eight OmniRats were immunized alternately with huCD33 ECD-HSA and cyno CD33 ECD-HSA. Following a 46 day immunization regimen, lymph nodes from all eight OmniRats were harvested and used to generate hybridomas. Eighty-one 96-well plates of hybridoma supernatants were screened via binding ELISA and AlphaLISA using standard techniques, of which 128 hybridoma supernatants were selected for specific binding to huCD33 ECD-HSA and cyno CD33 ECD-HSA. Most of the 128 supernatants were also positive for binding to cells over-expressing huCD33 or cyCD33.

Six additional OmniRats were immunized with rhuCD33 only. Following a 31 day immunization regimen, lymph nodes from all six OmniRats were harvested and used to generate hybridomas. Thirty 96-well plates of hybridoma supernatants were screened via binding ELISA using standard techniques, of which 94 hybridoma supernatants were selected for specific binding to huCD33 ECD-HSA and cyno CD33 ECD-HSA. Hybridoma lysates were prepared from the positive clones and progressed to v region cloning described below.

OmniMouse

A human immunoglobulin transgenic mouse strain (OmniMouse®; Ligand Pharmaceuticals) was used to develop human CD33 monoclonal antibody expressing hybridoma cells. The OmniMouse® contains chimeric human/rat IgH loci together with fully human IgL loci. The mice exhibit reduced expression of mouse immunoglobulin, and in response to immunization, the introduced human heavy and light chain transgenes undergo class switching and somatic mutation to generate high affinity chimeric human/rat IgG monoclonal antibodies with fully human variable regions.

When immunized with recombinant human and cynomolgus CD33 (huCD33 ECD-HSA and cyno CD33 ECD-HAS respectively), this transgenic mouse produces chimeric human/rat IgG antibodies to human CD33, some of which also bind to cynomolgus CD33.

Four OmniMice were immunized alternately with huCD33 ECD-HSA and cyno CD33 ECD-HSA. Following a 53 day immunization regimen, spleens and lymph nodes from all four OmniMice were harvested and used to generate hybridomas. Forty-eight 96-well plates of hybridoma supernatants were screened via binding ELISA and AlphaLISA, of which 8 hybridoma supernatants were selected for specific binding to huCD33 ECD-HSA and cyno CD33 ECD-HSA. Hybridoma lysates were prepared from the positive clones and progressed to v region cloning described below.

V Region Cloning

Total RNA from hybridoma cell lysates was purified using RNeasy 96 kit (Qiagen; Hilden, Germany) following the manufacturer's protocol, and the resulting RNA was quantitated using Drop Sense and stored at −80° C. or cDNA was synthesized using Invitrogen SuperScript III First-Strand Synthesis System for RT-PCR (Invitrogen; Carlsbad, CA). The first strand cDNA Synthesis was carried out using gene specific primers annealed to the constant regions of heavy, kappa, and lambda chains, respectively. The RT-PCR reaction mixture is comprised of up to 3 μg of purified RNA, gene specific primer, dNTP mix, reaction buffer, 25 mM MgCl₂, DTT, RNaseOUT™ (40 U/μl, Invitrogen), and SuperScript™ III RT (200 U/μl, Invitrogen Cat #18080-051), and incubate at 50° C. for 50 minutes and 85° C. for 5 minutes. The resulting single-stranded cDNA was stored at −20° C., or the single-stranded DNA was PCR amplified. The PCR reaction was carried out using Platinum Pfx polymerase (Invitrogen). The v-region fragments were amplified by forward and reverse primers annealing to the leader sequences and constant regions of heavy, kappa and lambda chains, respectively, using optimized PCR conditions. The resulting PCR fragments were run on the gel and sequenced using pre-designed primers to obtain v-region sequences. The resulting .abi files of v-region sequences were collected and analyzed by the Sanger v-region sequence analysis program created at Janssen Biologics Discovery. The AA sequences of the recovered v-regions were registered in the internal database, codon optimized and cloned into the pUnder-based expression vector carrying the appropriate constant region of the desired human antibody isotype: IgG1 F405L and IgG4 PAA. A total of 76 OMNIRat antibodies and 8 OMNIMouse antibodies were successfully cloned and proceeded for further characterization. The tables below summarize the sequences from the top 32 identified in the OMNIRat campaigns (see Table 41) and the 8 identified in the OMNIMouse campaign (see Table 42) with several of the OMNIRat antibodies cloned into IgG1 as well as IgG4 PAA and all from the OMNIMouse campaign were cloned into both IgG1 and IgG 4 PAA.

TABLE 41

Antibody sequences identified via

CD33 immunization in OMNIRat

mAb
HC ID
HC Isotype
LC ID

C33B46
C33H108
hulgG1F405L
C33L74

C33B48
C33H80
hulgG1F405L
C33L73

C33B52
C33H42
hulgG1F405L
C33L8

C33B54
C33H44
hulgG1F405L
C33L10

C33B55
C33H45
hulgG1F405L
C33L11

C33B56
C33H46
hulgG1F405L
IAPL24

C33B61
C33H48
hulgG1F405L
C33L58

C33B62
C33H49
hulgG1F405L
C33L59

C33B63
C33H51
hulgG1F405L
C33L34

C33B64
C33H52
hulgG1F405L
N46L109

C33B66
C33H55
hulgG1F405L
C33L42

C33B72
C33H65
hulgG1F405L
C33L47

C33B73
C33H66
hulgG1F405L
C33L60

C33B75
C33H70
hulgG1F405L
N46L109

C33B77
C33H72
hulgG1F405L
C33L40

C33B79
C33H74
hulgG1F405L
C33L38

C33B80
C33H76
hulgG1F405L
C33L39

C33B82
C33H78
hulgG1F405L
C33L57

C33B83
C33H81
hulgG1F405L
C33L53

C33B87
C33H87
hulgG1F405L
C33L35

C33B88
C33H88
hulgG1F405L
C33L61

C33B89
C33H90
hulgG1F405L
C33L51

C33B94
C33H98
hulgG1F405L
C33L69

C33B95
C33H98
hulgG1F405L
IAPL24

C33B96
C33H99
hulgG1F405L
C33L37

C33B101
C33H69
hulgG1F405L
C4LL152

C33B107
C33H68
hulgG1F405L
C33L17

C33B120
C33H87
hulgG1F405L
C33L41

C33B122
C33H92
hulgG1F405L
C33L30

C33B123
C33H91
hulgG1F405L
C33L44

C33B124
C33H73
hulgG1F405L
C33L32

C33B125
C33H84
hulgG1F405L
C33L66

C33B760
C33H45
huIgG4 PAA
C33L11

C33B777
C33H65
huIgG4 PAA
C33L47

C33B778
C33H66
huIgG4 PAA
C33L60

C33B782
C33H72
huIgG4 PAA
C33L40

C33B792
C33H87
huIgG4 PAA
C33L35

C33B799
C33H98
huIgG4 PAA
C33L69

C33B806
C33H69
huIgG4 PAA
C4LL152

C33B830
C33H84
huIgG4 PAA
C33L66

C33B836
C33H80
huIgG4 PAA
C33L73

C33B937
C33H66
huIgG4 PAA
C33L132

HC: Heavy Chain;

LC: Light Chain

TABLE 42

Antibody sequences identified via

CD33 immunization in OMNIMouse

mAb
HC ID
LC ID

C33B901
C33H249
C33L115

C33B902
C33H250
C33L116

C33B903
C33H251
C33L117

C33B904
C33H252
C33L118

C33B905
C33H253
C33L119

C33B906
C33H254
C33L120

C33B907
C33H255
C33L121

C33B908
C33H256
C33L122

C33B909
C33H249
C33L115

C33B910
C33H250
C33L116

C33B911
C33H251
C33L117

C33B912
C33H252
C33L118

C33B913
C33H253
C33L119

C33B914
C33H254
C33L120

C33B915
C33H255
C33L121

C33B916
C33H256
C33L122

HC: Heavy Chain;

LC: Light Chain

5-4 Expi293 Small Scale Transfection and Purification

Antibodies identified in the immunization campaigns and subsequent v region cloning (into IgG1 F405L and IgG4 PAA) were expressed and purified via small 2 ml scale. Expi293™ cells (ThermoFisher Scientific) were seeded at 1.25×10⁵-2.25×10⁵viable cells/mL density in Expi293™ Expression Medium and cultured in polycarbonate, disposable, sterile, vented, non-baffled Erlenmeyer shake flasks in a 37° C., 7% CO2 shaker incubator (INFORS HT Multitron Pro). For routine cell growth in 125 mL-2 L shake flasks, the shake speed was set to 130 rpm for shakers with a 19 mm shaking diameter. Cells were sub-cultured when density reached log phase growth at 3×10⁶-5×10⁶viable cells/mL with a 98-99% viability.

On day of transfection, the viable cell density and percent viability was determined. Cells were transfected at a density of 3×10⁶viable cells/mL. For optimal transfection, sterile Heavy and Light Chain plasmid DNA at 0.1 mg/mL concentration in TE buffer (10 mM Tris-HC1, 1 mM EDTA, pH 8.0) is used.

Expi293™ cells were transfected following manufacturer's Transfection protocol (ThermoFisher Publication Number MAN0007814). Transfection was performed in 24-well deepwell plates (GE Healthcare). Briefly, plasmid DNA was diluted with 0.1 mL OptiMEM™ medium (ThermoFisher Scientific) at the following ratio: 0.250 μg Heavy Chain DNA: 0.750 Light Chain DNA: 0.5 μg pAdvantage. 5 μL of ExpiFectamine™ 293 Transfection Reagent was diluted and mixed gently with 954, OptiMEM™ medium and incubated for 1 min. The diluted ExpiFectamine™ 293 Reagent was added to the diluted DNA, mixed gently and the ExpiFectamine™ 293/plasmid DNA complexes were incubated at room temperature for 40 minutes. Post-incubation, 1.8 mL Expi293™ cells were added to the complexes incubated overnight in a 37° C., 7% CO2 shaker incubator.

On Day 1 post-transfection, 104, ExpiFectamine™ 293 Enhancer land 1004, Expifectamine 293™ Enhancer 2 were added and the plates were returned to the incubator for an additional 5 days. The culture was harvested on day 6 post-transfection by centrifugation at 850×G for 15 minutes before purification.

1.7 mls of clarified expression supernatants prepared above were transferred to a new 96 2 ml deepwell plate. Purification plates were prepared by pipetting 800 μl of a 1:4 mix of MabSelect SuRe (GE Healthcare) and DPBS −/− slurry into every well of a 96 well Acroprep Advance 1 μm glass filter plate (Pall). 200 mbar of vacuum pressure was applied to the plate to remove excess PBS and subsequently washed with 800 μl fresh PBS. 200 mbar vacuum pressure was applied to remove the wash buffer. The clarified supernatants were then transferred to the PBS washed resin, mixed gently and incubated for 15 minutes. Following the incubation, 200 mbar vacuum pressure was applied to remove the supernatant. The MabSelect SuRe was washed three times with PBS and once with 25 mM Sodium Acetate, pH 5 (TEKNOVA; Hollister, CA) with 200 mbar vacuum pressure applied between washes to remove excess buffer. mAbs bound to the resin were eluted using 0.1 M Sodium Acetate, pH 3.5 and incubated for 10 minutes for effective dissociation. The filter plate was placed atop a 96 deepwell plate and the eluted mAbs were collected in the bottom plate via centrifugation at 1000 g for 2 minutes. 80 μl of 2.5 M Tris-Actetate, pH 7.2 was added to neutralize the mAbs. The mAbs were dialyzed into PBS overnight in a 96 well DispoDIALYZER plate (Harvard Apparatus; Holliston, MA), transferred to a 96 well Acroprep Advance 0.2 □m Supor filter plate (Pall; Port Washington, NY), placed atop a 96 deepwell plate and the protein solutions filtered via centrifugation at 1,500 g for 15 minutes in a desktop centrifuge. Protein concentrations were determined by A280 measurement on the filtrate using a DropSense Instrument (Trinean).

TABLE 43

CD33 mAb Heavy Chain and Light Chain Variable Regions

SEQ

ID

NO
AMINO ACID SEQUENCE

HC ID

B23H1
246
QITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGMGVSWIRQPPGKALEWLAHIYWDDDKRYNPS

LKSRLTITKDTSKNQVVLTMTNMDPVDTATYYCARLYGFTYGFAYWGQGTLVTVSS

CD3H141
247
EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATY

YAASVKGRFTISRDDSKNSLYLQMNSLKTEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVT

VSS

CD3H219
20
QVQLQQSGPRLVRPSQTLSLTCAISGDSVFNNNAAWSWIRQSPSRGLEWLGRTYYRSKWLYD

YAVSVKSRITVNPDTSRNQFTLQLNSVTPEDTALYYCARGYSSSFDYWGQGTLVTVSS

C33H42
248
QLQLQESGPGLVNPSETLSHTCTVSGGSISSSSHYWGWIRQPPGKGLEWIGKIYYSGNTYYNPS

LKSRVTISIDTSKNQFSLKMSSVTAADTAVYYCARLADVVVVPAARYFDSWGQGTLVTVSS

C33H44
249
QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWYND

YAVSVRSRITINPDTSKNQFSLQLNSVTPEDTAVYHCARETMFRGLMDYWGQGTLVTVSS

C33H45
250
QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQSPGKGLEWVAVISYDGSNKYCAD

SVKGRFTISRDNSKSTLYLQMNSLRAEDTAVYYCAKDFRSLDWLPPDSTSYDGMDVWGQGTT

VTVSS

C33H46
251
QVQLVQSGSELKKPGASVKVSCKASGYTFTNYAMNWVRQAPGQGLEWMGWINTNTGNPTY

AQAFTGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCARDREVRDYWGQGTLVTVSS

C33H48
252
QLQLQESGPGLVKPSETLSLTCTVSGGSIRSTNYYWGWIRQPPGKGLEWIGTIYYSGNTYYNPS

LKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARLADVVVVPAARYFDYWGQGILVTVSS

C33H49
253
QLQLQESGPGLVKPSETLSLTCTVSGGSIRSSGFYWGWIRQPPRKGLEWIGTIYYSGNTYYNPSL

KSRVTISVDTSKNQFSLKLSSVTAADTAVYFCARLADVVVVPAARYFDNWGQGTLVTVSS

C33H51
254
QLQLQESGPGLVKPSETLSLTCTVSGGSISTGRYYWGWIRQPPGKGVIWIGNIYYSGNTYYNPS

LKSRVTISVDTSKNQFSLKLNSVTAADTAVYYCARLGSLVVVPAAMSFDYWGQGTLVTVSS

C33H52
255
QLQLQESGPGLVKPSETLSLTCTVSGGSIRGSSYYWGWVRQPPGKGLEWIGSIYSSGNTYYNPS

LKSRVTISVDTSKNQFSLKLSSVTAADTALYYCARLGSLVVVPAAMSFDYWGQGTLVTVSS

C33H55
256
QVQLQESGPGLVKPSGTLSLTCAVSGGSISSSNWWSWVRQPPGRGLEWIGEIYHSGNTNNSPSL

KSRVTISADKSKNQFSLKLSSVTAADTAVYFCARIIAVARYFDSWGQGTLVTVSS

C33H65
257
QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVVVISYDGSNKYYA

DSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDFRDFDWLPPDSTSYHGMDVWGQG

TTVTVSS

C33H66
258
QVQLVESGGGVVQPGRSLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVAVISYDGSNKYYA

DSVKGRFTISRDNSKNTLYLQMNSLRAEGTAVYYCAKDFRSFDWLPPDSASYHGMDVWGQG

TTVTVSS

C33H68
259
EVQLLESGGGLVQPGGSLGLSCAASGFTFSGYAMSWVRQAPGKGLNWVSAIDYSGNDTYYAD

SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKESQLLHGLFEHWGQGILVTVSS

C33H69
26
QLQLQESGPGLVKPSETLSLTCTVSGGSISSSSYYWGWIRQPPGKGLDWIGSINYSGSTYYNPSL

KSRVTISVDTSKIQFSLKLRSVTAADTAVYYCARLDGYESPFDYWGQGTLVTVSS

C33H70
261
QLQLQESGPGLVKPSETLSLTCTVSGGSIRGSSYYWGWIRQPPGKGLEWIGSIYSSGNTYYNPSL

KSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARLGSLVVVPAAMSFDYWGQGTLVTVSS

C33H72
262
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVANIKQHGSEKYYV

DSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDRDLGYFDYWGQGTLVTVSS

C33H73
263
EVQLLESGGGLVQPGGSLRLSCAASRFTFSSYAMTWVRQAPGKGLEWVSTINISGGSTYYADS

VKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTKGGYSSGPFDYWGQGTLVSVSS

C33H74
264
QVQLVESGGGVVQPGRSLRLSCAASRFTFSSYGMHWVRQAPGKGLEWVAVISYDGSNKYYA

DSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVHYCAKDFRSFDWLPPDSASYHGMDVWGQG

TTVTVSS

C33H76
265
EVQLLESGGGLVQPGGSLRLSCAASGFTFNYAMSWVRQAPGKGLEWVSAISGSGGSTYYADS

VKGRFTISRDISKNTLYLQMNSLRAEDTAVYYCARTYNSGYYDGDFDYWGQGTLVTVSS

C33H78
266
QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYDGSNKYYA

DSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCAKDFRYFDWLPPDSSSYYGMDVWGQGT

TVTVSS

C33H80
267
QVQLVQSGSELRKPGASVKVSCKASGYTFTNYAMNWVRQAPGQGLEWMGWINTNTGNPTY

AQGFTGRFVFSLDTSVSSAYLQISSLKAEDTAMYYCATDRDRGTDYWGQGTLVTVSS

C33H81
268
QVQLVESGGGVVQPGRSLRLSCAASGFTFSAYGMHWVRQAPGKGLEWVAVISYDGSNKYYA

DSVKGRFTISRDNSKNTLYLQMNSLRAEGTAVYYCAKDFRSFDWLPPDSASYHGMDVWGQG

TTVTVSS

C33H84
269
QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYDGSNKYYA

DSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDFRSFDWLPPDSTSYYGMDVWGQGT

TVTVSS

C33H87
270
EVQLVESGGGFVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVANIKQHGSEKYYV

DSVKGRFTISRDNVKNSLYLQMNSLRTEDTAVYYCARDRDLGYFDYWGQGTLVTVSS

C33H88
271
QVQLVQSGSELKKPGASVKVSCKASGYTLTRSAMNWVRQAPGQGLEWMGWINTNTGNPTYA

QGFTGRFVFSLDTSVNTAYLLISSLKTEDTAVYYCASDILPGYHEDYWGQGTLVTVSS

C33H90
272
QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWYND

YALSVQSRITINPDTSKNQFSLQLNSVTPEDTAVYYCAREVAVAASFDYWGQGTLVTVSS

C33H91
273
QLQLQESGPGLVKPSETLSLTCTVSGGSISSRSHYWGWIRQPPGVGLEWIGSIYYTGSTYYNPSL

KSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARLADIVVVPAARYFDYWGQGTLVTVSS

C33H92
274
QLQLQESGPGLVKPSETLSLTCTVSGGSIRSSSYYWGWIRQPPGKGPEWIGSIYSSGNTYYNPSL

KSRVTISVDTSKNQFSLKLISMTAADTAVFYCARLAATIVVPAARYFDCWGQGTLVTVSS

C33H98
275
EVQLVESGGGFVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVANIKQHGSEKYYV

DSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDRDLGYFDYWGQGTLVTVSS

C33H99
276
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMTWVRPAPGKGLEWVANIKRDGGEKYYV

DSVKGRFTISRDNAANSLYLQMNSLRVEDTAVYYCARPFYDHFDYWGQGTLVTVSS

C33H108
277
QVQLVQSGSELKKPGASVKVSCKASGYTFSTYAMNWVRQAPGQGLEWMGWINTNTGNPTYA

QGFTGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCARDRDRGTDYWGQGTLVTVSS

C33H249
278
EVQLVESGGGLVQPGRSLRLSCVASGFTFDDYAIHWVRQAPGKGLEWVSGLSWNGGNIGYAD

SVKGRFTISRDNAKNSLYLQMNSLKTEDTAFYYCTKDTPYGDYFDYWGQGTLVTVSS

C33H250
279
EVQLVESGGGLVQPGRSLRLSCAGSGFTFDDYAIHWVRQAPGKGLEWVSGLSWNGGNIGYAD

SVKGRFTISRDNAKNSLYLQLNSLKTEDTAFYYCAKDSPYGDYFDYWGQGTLVTVSS

C33H251
280
EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGIGWSGGSIVYAD

SVKGRFKISRDNAKNSLYLQMNSLRAEDTALYYCAKDSPYGDFFDYWGQGTLVTVSS

C33H252
281
EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGIGWSGGSIVYAD

SVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCAKDSPYGDFFDYWGQGTLVTVSS

C33H253
282
EVQLLESGGGLVQPGGSLKLSCTASGFTFRSYAMSWVRQAPGKGLEWVSAINGYGDGRYYAD

SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYSCAKDQGFGELFFDYWGQGTLVTVSS

C33H254
283
QVQLVESGGGVVQPGRSLRLSCAASGFTFSYYGMHWVRQAPDKGLEWVAVIWFDGNNKYY

ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDRELLFDYWGQGTLVTVSS

C33H255
284
EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQVPGEGLEWVSGISWNGGDMVYA

DSVKGRFTISRDNAKNSLYLQMNSLRPEDTALYYCVKDMPYFDFLTGSDYYYYGMDVWGQG

TTVTVSS

C33H256
285
QVQLVESGGGVVQPGRSLRLSCATSGFTFSNYGMHWVRQAPGKGLEWVAVIWYVGSHKYYA

DSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDGSLCFDYWGQGTLVTVSS

LC ID

B23L3
286
DIVMTQSPDSLAVSLGERATINCRASQSVDYNGISYMHWYQQKPGQPPKLLIYAASNPESGVP

DRFSGSGSGTDFTLTISSLQAEDVAVYYCQQIIEDPWTFGQGTKVEIK

CD3L66
287
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGTPAR

FSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKLTVL

CD3L150
52
QSALTQPASVSGSPGQSITISCTGTSSNIGTYKFVSWYQQHPDKAPKVLLYEVSKRPSGVSSRFS

GSKSGNTASLTISGLQAEDQADYHCVSYAGSGTLLFGGGTKLTVL

C33L8
288
SYELTQPPSVSVSPGQTASIICSGDKLGNKYACWYQQKPGQSPVLVIYQDSKRPSGIPERFSGSN

SGNTATLTISGTQAVDEADYYCQAWDSSTYVFGTGTKVTVL

C33L10
289
SYVLTQPPSVSVAPGQTARITCGGSNIGSKSVHWYQQKPGQAPVMVVYDDSDRPSGIPERFSGS

NSGNTATLTISRVEAGDEADYYCQVWDSSSDVVFGGGTKLTVL

C33L11
290
SYELTQPPSVSVSPGQTASITCSGHKLGDKYACWYQQKPGQSPVVVIYKDSKRPSGIPERFSGS

NFGNTATLTISGTQAMDEADYYCQAWDSSTVVFGGGTKLTVL

IAPL24
291
SYELTQPPSVSVSPGQTASITCSGDKLGDKYACWYQQKPGQSPVLVIYQDSKRPSGIPERFSGSN

SGNTATLTISGTQAMDEADYYCQAWDSSTVVFGGGTKLTVL

C33L58
292
SYELTQPPSVSVSPGQTASITCSGDKLGDKYACWYQQKPGQSPVLVIYQDYKRPSGIPERFSGS

NSGNTATLTISGTQAMDEADYYCQAWDSSTYVFGTGTKVTVL

C33L59
293
SYELTQPPSVSVSPGQTASITCSGDKLGDKYACWYQQKPGQSPVLVIYQDYKRPSGIPERFSGS

NSGNTATLTISGTQTMDEADYYCQAWDISTYVFGTGTKVTVL

C33L34
294
SYELTQPPSVSVSPGQTASITCSGDKLGDKYACWYQLRPGQSPILVIYQDSNRPSGIPERFSGSNS

GNTATLTISGTQAMDEADYYCQAWDSSTWVFGGGTKLTVL

N46L109
295
SYELTQPPSVSVSPGQTASITCSGDKLGDKYACWYQQKPGQSPVLVIYQDSKRPSGIPERFSGSN

SGNTATLTISGTQAMDEADYYCQAWDSSTWVFGGGTKLTVL

C33L42
296
SYVLTQPPSVSVAPGQTARITCGGNNIGEKSVHWYQQKPGQAPVLVVYDDSDRPPGIPERFSGS

NSGNTATLTITRVEAGDEADYYCQVWDSSSDHVVFGGGTKLTVL

C33L47
297
SYELTQPPSVSVSPGQTASITCSGDKLGDKYACWYQQKPGQSPVVVIYQDRKRPSGIPERFSGS

NFGNTATLTISGTQAMDEADYYCQAWDSSTVVFGGGTKLTVL

C33L60
298
SYELTQPPSVSVSPGQTASITCSGDKLGDKYACWYQQKPGQSPVLVIYQDGKRPSGIPERFSGS

NFGNKATLTISGTQAMDEADYYCQAWDRNTVVFGGGTKLTVL

C33L17
299
DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKAGQPPKLLIYWASTRESG

VPDRFSGSGSGTDFILIISSLQAEDVAVYYCQQYYGTPWTFGQGTKVEIK

C4LL152
300
DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSG

SGSGTDFTLTISSLQPEDFATYYCQQANSFPFTFGPGTKVDIK

C33L40
301
SYELTQPPSVSVSPGQTASITCSGNKLGAKFASWYQQKPGQSPVLVIYQDNKRPSGIPERFSGSN

SGNTATLTISGTQAVDEADYYCQAWDSSTVVFGGGTKLTVL

C33L32
302
SYELTQPPSVSVSPGQTASITCSGDKLGDKYVRWYQQKTGQSPVLVMYQDSKRPSGIRERFYG

SNSGNTATPTISGTQAVDEAEYYCQAWDSSTGVVFGGGTKLTVL

C33L38
303
SYELTQPPSVSVPPGQTASITCSGDKLGDKYACWYQQKPGQSPVLVIYQDNKRPSGIPERFSGS

NSGNTATLTISGTQAMDEADYYCQAWGRNTVVFGGGTKLTVL

C33L39
304
QSALTQPASVSGSPGQSIPISSTGTSSDDGKNNIVSWYQQHPGKAPKLMIYKDSKRPSGVSNRFS

GSKSGNTASLTISGLQADDEADYHCCSYAGASNHVVFGGGTKLTVL

C33L57
305
SYELTQPPSVSVSPGQTASITCSGDELGNKYACWYQQKPGQSPVVVVYQDRKRPSGIPERFSGS

NFGNTATLTISGTQAMDEADYYCQAWDSSTVVFGGGTKLTVL

C33L73
306
QSALTQPASVSGSPGQSITISCTGTSSDVGDYNYVSWYQQHPGKVPKLMIYDVSNRPSGVSNRF

SGSMSGNTASLTISGLQAEDEADYYCSSYSSSSALEVFGGGTKLTVL

C33L53
307
SYELTQPPSVSVSPGQTASITCSGDKLGDKYACWYQQKPGQSPVLVIYQDNKRPSGIPERFSGS

NSGNTATLTISGTQAMDEADYYCQAWDSNTVVFGGGTKLTVL

C33L66
308
SYELTQPPSVSVSPGQTASITCSGDKLGDKYVCWYQQKPGQSPVVVIHQDRKRPSGIPERFSGS

NFGNTATLTISGTQAMDEADYYCQAWDSSTVVFGGGTKLTVL

C33L35
309
SYELTQPPSVSVSPGQTASITCSGDKLGNKYASWYQQKPGQSPVLVIYQDTKRPSGIPERVSGS

NSGNTATLTISGTQAMDEADYHCQAWDSSTVVFGGGTKLTVL

C33L61
310
QSALTQPASVSGSPGQSITISCTGINSDVGSYDLVSWYQQHPGKAPKLLIYDGSERPSGVFGRFS

GSKSDNTTSLTISGLQAEDEAAYYCCSYEVTTTYVVFGGGTKLTVL

C33L51
311
SYVLTQPPSVSVAPGQTARITCGGNNIGSKSVHWSQQKPGQAPVLVVYDDSDRPSGIPERFSGS

NSGNTATLTISRVEAGDEADYYCQVWDSNSDHVVFGGGTKLTVL

C33L44
312
SYELTQPPSVSVSPGQTASITCSGDKLGDKYACWYQQKPGQSPVLVIYQDSNRPSGIPERFSGSN

SGNTATLTISETQAMDEADYYCQAWDSSTYVFGTGTKVTVL

C33L30
313
SYELTQPPSVSVSPGQTVSISCSGDRLGDKYACWYQQKPGQSPVLVIYQDSKRPSGIPERFSGSN

SGNTATLTISGTQAMDEADYYCQAWDSSSYVFGTGTKVTVL

C33L69
314
SYELTQPPSVSVSPGQTASITCSGDKLGSKFACWYQQKPGQSPVLVIYQDSKRPSGIPERFSGSN

SGNTATLTISGTQAMDEADYYCQAWDSSTVVFGGGTKLTVL

C33L37
315
SYVLTQPPSVAVAPGQTARITCGGSNIGKISVHWYQQKAGQAPVLVVHDDRARPSGIPERLSGS

NSGTTATLTISRVEVGDEADYYCQVWNSSSVHPVFGGGTKLTVL

C33L74
316
QSALTQPASVSGSPGQSITISCTGTSSDVGDDNYVSWYQQHPGKAPKLMIYDVSNRPSGVSNRF

SGSKSGNTASLTISGLQSEDEADYYCSSYSSSTTLEVFGGGTKLTVL

C33L115
317
DIQMTQSPSSVWASVGDRVTITCRASQGISSWLAWYQQQPGKAPNLLIYRSSSLQSGVPSRFSG

SGSGTDFTLTISSLQPEDFATYYCQQDNSFPYTFGQGTKLEIK

C33L116
318
DIQMTQSPSSEWASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIYGASSWQSGVPSRFS

GSGSGTDFTLTISSLQPEDFATYYCQQDNSFPYTFGQGTKLEIK

C33L117
319
DIVMTQSPDSLAVSLGERATINCKSSQTVLYSSNNKNYLAWYQQKPGQPPKLLISWASTRKSG

VPDRFSGSGSGTDFTLTVSSLQAEDVAVYYCQHYYSTPYTFGQGTKLEIK

C33L118
320
DIVMTQSPDSLAVSLGERATINCKSSQTVFYSSNNKNYLAWYQQKPGQPPKLLISWASTRKSG

VPDRFSGSGSGTDFTLTVSSLQAEDVAVYYCQHYYSTPYTFGQGTKLEIK

C33L119
321
DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKASSLESGVPSRFSGS

GSGTEFTLTISSLQPDDFATYCCQQYNSYPWTFGQGTKVEEK

C33L120
322
SYELTQPPSVSVSPGQTASITCSGDELGDMYACWYQQKPGQSPLVVIYQDSKRPSGIPERFSGSN

SGNTATLTISGTQAMDEAAYYCQTWDTRIAVFGGGTNLTVL

C33L121
323
SYELTQPPSVSVSPGQTASITCSGDNLGNEHVCWYHQKPGQSPVLVIYQNNKRPSGIPERFSGSN

SGNTATLSISGTQATDEADYYCQAWDSTTAVFGGGTKLTVL

C33L122
324
SYELTQPPSVSVSPGQTANISCSGVTLGYNYAYWYQQKPGQSPILVISQDTQRPSGIPERFSGSN

SGNTATLTISGTQAMDEAAYYCQAWDITTVLFGGGTKLTVL

C33L132
325
SYELTQPPSVSVSPGQTASITCSGDKLGDKYASWYQQKPGQSPVLVIYQDGKRPSGIPERFSGSN

FGNKATLTISGTQAMDEADYYCQAWDRNTVVFGGGTKLTVL

C33L41
326
SYELTQPPSVSVSPGQTASITCSGDKLGNKYASWYQQKPGQSPVLVIYQDSKRPSGIPERFSGSN

SGNTATLTISGTQAMDEADYYCQAWDSSTVVFGGGTKLTVL

TABLE 44

CD33 mAb Heavy Chain CDR1-3 sequences

HC ID
CDR1
ID
CDR2
ID
CDR3
ID

B23H1
GFSLSTSGMG
327
IYWDDDK
368
ARLYGFTYGFAY
409

CD3H141
GFTFNTYA
328
IRSKYNN
369
ARHGNFGNSYVS
410

YAT

WFAY

CD3H219
GDSVFNNNAA
329
TYYRSKWL
370
ARGYSSSFDY
411

Y

C33H42
GGSISSSSHY
330
IYYSGNT
371
ARLADVVVVPAA
412

RYFDS

C33H44
GDSVSSNSAA
331
TYYRSKWY
372
ARETMFRGLMDY
413

N

C33H45
GFTFSSYG
332
ISYDGSNK
373
AKDFRSLDWLPP
414

DSTSYDGMDV

C33H46
GYTFTNYA
333
INTNTGNP
374
ARDREVRDY
415

C33H48
GGSIRSTNYY
334
IYYSGNT
375
ARLADVVVVPAA
416

RYFDY

C33H49
GGSIRSSGFY
335
IYYSGNT
376
ARLADVVVVPAA
417

RYFDN

C33H51
GGSISTGRYY
336
IYYSGNT
377
ARLGSLVVVPAA
418

MSFDY

C33H52
GGSIRGSSYY
337
IYSSGNT
378
ARLGSLVVVPAA
419

MSFDY

C33H55
GGSISSSNW
338
IYHSGNT
379
ARIIAVARYFDS
420

C33H65
GFTFSSYG
339
ISYDGSNK
380
AKDFRDFDWLPP
421

DSTSYHGMDV

C33H66
GFTFSSYG
340
ISYDGSNK
381
AKDFRSFDWLPP
422

DSASYHGMDV

C33H68
GFTFSGYA
341
IDYSGNDT
382
AKESQLLHGLFE
423

H

C33H69
GGSISSSSYY
342
INYSGST
383
ARLDGYESPFDY
424

C33H70
GGSIRGSSYY
343
IYSSGNT
384
ARLGSLVVVPAA
425

MSFDY

C33H72
GFTFSSYW
344
IKQHGSEK
385
ARDRDLGYFDY
426

C33H73
RFTFSSYA
345
INISGGST
386
TKGGYSSGPFDY
427

C33H74
RFTFSSYG
346
ISYDGSNK
387
AKDFRSFDWLPP
428

DSASYHGMDV

C33H76
GFTFNYA
347
ISGSGGST
388
ARTYNSGYYDGD
429

FDY

C33H78
GFTFSSYG
348
ISYDGSNK
389
AKDFRYFDWLPP
430

DSSSYYGMDV

C33H80
GYTFTNYA
349
INTNTGNP
390
ATDRDRGTDY
431

C33H81
GFTFSAYG
350
ISYDGSNK
391
AKDFRSFDWLPP
432

DSASYHGMDV

C33H84
GFTFSSYG
351
ISYDGSNK
392
AKDFRSFDWLPP
433

DSTSYYGMDV

C33H87
GFTFSSYW
352
IKQHGSEK
393
ARDRDLGYFDY
434

C33H88
GYTLTRSA
353
INTNTGNP
394
ASDILPGYHEDY
435

C33H90
GDSVSSNSAA
354
TYYRSKWY
395
AREVAVAASFDY
436

N

C33H91
GGSISSRSHY
355
IYYTGST
396
ARLADIVVVPAA
437

RYFDY

C33H92
GGSIRSSSYY
356
IYSSGNT
397
ARLAATIVVPAA
438

RYFDC

C33H98
GFTFSSYW
357
IKQHGSEK
398
ARDRDLGYFDY
439

C33H99
GFTFSSYW
358
IKRDGGEK
399
ARPFYDHFDY
440

C33H108
GYTFSTYA
359
INTNTGNP
400
ARDRDRGTDY
441

C33H249
GFTFDDYA
360
LSWNGGNI
401
TKDTPYGDYFDY
442

C33H250
GFTFDDYA
361
LSWNGGNI
402
AKDSPYGDYFDY
443

C33H251
GFTFDDYA
362
IGWSGGSI
403
AKDSPYGDFFDY
444

C33H252
GFTFDDYA
363
IGWSGGSI
404
AKDSPYGDFFDY
445

C33H253
GFTFRSYA
364
INGYGDGR
405
AKDQGFGELFFD
446

Y

C33H254
GFTFSYYG
365
IWFDGNNK
406
ARDRELLFDY
447

C33H255
GFTFDDYA
366
ISWNGGDM
407
VKDMPYFDFLTG
448

SDYYYYGMDV

C33H256
GFTFSNYG
367
IWYVGSHK
408
ARDGSLCFDY
449

TABLE 45

CD3 mAb Light Chain CDR1-3 sequences

LC ID
CDRI
ID
CDR2
ID
CDR3
ID

B23L3
QSVDYNGISY
450
AAS
492
QQIIEDPWT
534

CD3L66
TGAVTTSNY
451
GTN
493
ALWYSNLWV
535

CD3L150
SSNIGTYKF
452
EVS
494
VSYAGSGTLL
536

C33L8
KLGNKY
453
QDS
495
QAWDSSTYV
537

C33L10
NIGSKS
454
DDS
496
QVWDSSSDVV
538

C33L11
KLGDKY
455
KDS
497
QAWDSSTVV
539

IAPL24
KLGDKY
456
QDS
498
QAWDSSTVV
540

C33L58
KLGDKY
457
QDY
499
QAWDSSTYV
541

C33L59
KLGDKY
458
QDY
500
QAWDISTYV
542

C33L34
KLGDKY
459
QDS
501
QAWDSSTWV
543

N46L109
KLGDKY
460
QDS
502
QAWDSSTWV
544

C33L42
NIGIKS
461
DDS
503
QVWDSSSDHVV
545

C33L47
KLGDKY
462
QDR
504
QAWDSSTVV
546

C33L60
KLGDKY
463
QDG
505
QAWDRNTVV
547

C33L17
QSVLYSSNNKNY
464
WAS
506
QQYYGTPWT
548

C4LL152
QGISSW
465
AAS
507
QQANSFPFT
549

C33L40
KLGAKF
466
QDN
508
QAWDSSTVV
550

C33L32
KLGDKY
467
QDS
509
QAWDSSTGVV
551

C33L38
KLGDKY
468
QDN
510
QAWGRNTVV
552

C33L39
SSDDGKNNI
469
KDS
511
CSYAGASNHVV
553

C33L57
ELGNKY
470
QDR
512
QAWDSSTVV
554

C33L73
SSDVGDYNY
471
DVS
513
SSYSSSSALEV
555

C33L53
KLGDKY
472
QDN
514
QAWDSNTVV
556

C33L66
KLGDKY
473
QDR
515
QAWDSSTVV
557

C33L35
KLGNKY
474
QDT
516
QAWDSSTVV
558

C33L61
NSDVGSYDL
475
DGS
517
CSYEVTTTYVV
559

C33L51
NIGSKS
476
DDS
518
QVWDSNSDHVV
560

C33L44
KLGDKY
477
QDS
519
QAWDSSTYV
561

C33L30
RLGDKY
478
QDS
520
QAWDSSSYV
562

C33L69
KLGSKF
479
QDS
521
QAWDSSTVV
563

C33L37
NIGKIS
480
DDR
522
QVWNSSSVHPV
564

C33L74
SSDVGDDNY
481
DVS
523
SSYSSSTTLEV
565

C33L115
QGISSW
482
RSS
524
QQDNSFPYT
566

C33L116
QGISSW
483
GAS
525
QQDNSFPYT
567

C33L117
QTVLYSSNNKNY
484
WAS
526
QHYYSTPYT
568

C33L118
QTVFYSSNNKNY
485
WAS
527
QHYYSTPYT
569

C33L119
QSISSW
486
KAS
528
QQYNSYPWT
570

C33L120
ELGDMY
487
QDS
529
QTWDTRIAV
571

C33L121
NLGNEH
488
QNN
530
QAWDSTTAV
572

C33L122
TLGYNY
489
QDT
531
QAWDITTVL
573

C33L132
KLGDKY
490
QDG
532
QAWDRNTVV
574

C33L41
KLGNKY
491
QDS
533
QAWDSSTVV
634

5-5 Characterization of Anti-CD33 mAbs

OMNIRat antibodies identified via immunization, v-region cloned and subsequently expressed and purified were characterized further for binding to CD33 expressing cells and binding to recombinant antigens. The purified antibodies were assessed for binding to stably transfected HEK293F cells expressing human CD33 or cyno CD33 (generation described above) along with the parental HEK293F as negative control. Cells were harvested from tissue culture flasks using non-enzymatic dissociation buffer (Thermo Scientific). The flasks were rinsed twice with PBS and dissociation buffer was added to the flask, and the flask was incubated for 10 minutes at 37° C. until the cells became non-adherent. The cells were centrifuged at 300 g for 5 minutes and resuspended at 1.0×10⁶cells/ml in staining buffer (Becton Dickinson; Franklin Lakes, NJ). 50,000 cells/well of each cell type was plated in 50 μl of staining buffer in round bottom plates (Becton Dickinson). 50 μl of 2× concentration test mAb or isotype control was added at 3 dilutions and zero (120 nM, 12 nM, and 1.2 nM and 0 nM), and the resultant solution was incubated 30 min at 4° C. 100 μl staining buffer was added to all wells of each plate, the plates were spun at 300 g for 5 min, the buffer was removed, 200 μl staining buffer was added to all wells of each plate, the plates were spun at 300 g for 5 min, and the buffer was removed. 50 μl of 2 μg/ml of Goat-anti-human Fc AF647 secondary antibody (Jackson Immunoresearch; West Grove, PA) was added to all wells of the plates, and the plates were incubated for 30 min at 4° C. 100 μl staining buffer was added to all wells of the plates, the plates were spun at 300 g for 5 min, and the buffer was removed. 200 μl running buffer (running buffer is Staining buffer, 1 mM EDTA, 0.1% Pluronic Acid) was added to all wells of the plates, the plates were spun at 300 g for 5 min, and the buffer was removed. 30 μl running buffer containing Sytox Green live/dead dye (ThermoFisher) was added to all wells with cells and the plates were read on an iQue IntelliCyt flow cytometer. Cells were gated on forward vs. side scatter to eliminate debris, then on singlets and then on live cells which excluded the Sytox stain. Antibody binding was assessed by the mean fluorescence intensity in the AF647 channel.

To begin assessing the biophysical binding properties of the purified mAbs an off-rate screen was performed. 76 OMNIRat anti-CD33 mAbs were tested for binding to recombinant human CD33 ECD-HSA (C33W2) and cyno CD33 ECD-HSA (C33W1) proteins (Janssen production) and the off-rate was measured by IBIS MX96 SPRi array platform (Carterra; Newton, PA). Goat anti-human Fc IgG (Jackson Immunoresearch, Cat #109-005-098) was directly immobilized via amine coupling at 100 μg/mL in acetate buffer, pH 4.5 using a CMD50m sensor chip (Xantec, lot CMD50m0415.a) with an association time of 10 minutes in the IBIS instrument. An average GAH-Fc immobilization level of ˜9000 Rus was achieved. The sensor chip was transferred to the Continuous Flow Microspotter (CFM) unit to capture each anti-CD33 mAb at 10 μg/ml for 10 minutes. Binding was measured on IBIS SPRi by single cycle kinetics without regeneration. Each antigen concentration series (3 μM in 3 fold dilution series) was sequentially injected from low (0.46 nM) to high concentrations (3 μM) to bind to captured mAbs with an association time of 5 minutes and dissociation time of 15 minutes using PBST (PBS with 0.005% Tween) as running buffer. The raw binding data (.trix file format) were referenced and aligned using SprintX software (Wasatch, Ver 1.9.3.2), then exported (.ibmx file format) to Scrubber software (Ver. 2.0) for 1:1 binding kinetic analyses (Wasatch, version 2.0.0.33) to extract the k_offresults.

Table 46 below summarizes the top 32 clones as assessed by binding to human and cyno CD33 expressing cell lines as well as towards recombinant antigen (off-rate of at least >10e-3 for one of the antigens). Of these 32, all but 4 showed appreciable binding to either human or cyno expressing cells. All 32 were carried for further characterization via epitope binning and full kinetic analysis.

TABLE 46

Cell Binding and Off-Rate analysis of the anti-CD33 antibodies derived from OMNIRat

Prot. AA ID
% Mon
60 nM CD33
6 nM CD33
0.6 nM CD33
0 nM CD33
K_D

C33B48
91.96
400995.84
428948.75
391157.69
91.12
5.47E−05

C33B73
100.00
201493.02
33443.28
4034.64
93.98
9.12E−05

C33B125
98.48
258779.13
79728.78
9203.75
78.26
1.54E−04

C33B55
96.39
188278.42
59155.10
7625.56
105.39
2.15E−04

C33B96
98.75
476040.28
475653.41
187925.80
55.23
2.28E−04

C33B124
100.00
798.33
126.37
90.26
172.03
2.38E−04

C33B72
96.94
328194.72
105474.59
12506.85
93.32
2.84E−04

C33B79
100.00
236644.03
41925.89
4988.81
77.78
3.28E−04

C33B77
92.11
241787.16
88691.05
11484.97
69.46
3.37E−04

C33B82
96.21
188508.56
41264.92
5033.60
73.44
3.41E−04

C33B87
100.00
242185.48
79532.87
12547.05
73.65
3.52E−04

C33B80
98.33
5799.64
409.97
114.93
88.88
3.84E−04

C33B101
96.91
268805.28
204984.16
35513.63
70.07
3.98E−04

C33B83
98.07
92956.55
7856.70
1020.48
87.37
4.61E−04

C33B46
95.81
509865.97
447627.97
418017.22
134.53
4.67E−04

C33B94
98.31
200142.00
93852.22
13274.87
89.59
5.38E−04

C33B88
98.36
393148.13
481100.91
274293.53
94.81
8.25E−04

C33B66
98.71
444680.31
313288.41
56628.04
129.73
8.59E−04

C33B120
97.63
190036.14
60357.11
7054.28
92.94
1.40E−03

C33B64
98.13
200158.36
54138.77
7556.04
114.85
1.71E−03

C33B52
96.76
196557.09
46286.13
6751.01
82.46
3.13E−03

C33B56
95.59
143.73
79.73
111.95
138.04
4.02E−03

C33B75
98.68
163795.25
29603.57
4517.81
95.94
4.16E−03

C33B107
96.90
375388.25
339798.53
161369.64
86.54
4.44E−03

C33B63
98.79
247758.77
62221.71
9671.48
86.34
4.57E−03

C33B95
97.77
154556.58
44354.07
6402.00
87.38
5.99E−03

C33B61
98.87
198777.34
38699.10
5308.45
79.84
6.71E−03

C33B89
100.00
315.38
119.12
65.61
70.94
8.11E−03

C33B122
98.49
259183.69
84281.03
14291.17
65.01
8.74E−03

C33B62
99.05
157786.36
37359.44
6092.03
75.00
1.00E−02

C33B123
95.08
224078.95
88155.99
8864.39
71.05
1.03E−02

C33B54
100.00
147753.30
27461.06
3766.69
61.26
2.48E−02

Prot. AA
60 nM Cyno
6 nM Cyno
0.6 nM Cyno
0 nM Cyno
Cyno CD33

ID
CD33
CD33
CD33
CD33
binding K_D

C33B48
56491.32
47326.85
43351.12
94.01
1.20E−04

C33B73
14799.14
6987.92
795.57
72.51
4.08E−04

C33B125
15603.45
11526.47
3458.27
70.22
3.51E−04

C33B55
16020.78
9994.42
2433.94
69.38
1.16E−04

C33B96
37273.19
20087.29
11574.59
86.31
8.19E−04

C33B124
593.00
132.19
77.26
98.41
4.77E−04

C33B72
19422.07
13975.14
3894.84
90.81
7.63E−04

C33B79
15538.97
6427.73
1082.85
63.59
6.82E−03

C33B77
17516.20
11665.49
3601.76
85.23
4.18E−04

C33B82
14269.38
6622.07
1540.09
84.24
6.70E−04

C33B87
19597.18
12652.44
3266.36
103.07
2.28E−04

C33B80
4612.58
248.60
108.93
82.38
2.66E−04

C33B101
48016.75
46115.96
17989.37
79.69
1.06E−04

C33B83
5304.40
687.44
159.37
87.35
2.17E−03

C33B46
49840.14
49816.36
49729.78
92.05
1.48E−04

C33B94
16126.84
10782.54
3183.70
87.82
5.37E−04

C33B88
50388.18
43928.95
43940.23
90.13
3.89E−04

C33B66
48905.04
49076.39
42160.22
77.96
9.33E−05

C33B120
13211.32
7865.37
2726.18
75.77
8.54E−04

C33B64
21109.59
9685.04
3102.56
99.82
1.21E−03

C33B52
12582.90
8444.39
2063.44
75.24
1.20E−03

C33B56
104.27
85.94
78.56
83.31
8.46E−04

C33B75
12194.41
5577.80
1709.40
124.32
1.20E−03

C33B107
50325.07
47810.05
36786.69
55.11
1.35E−04

C33B63
18322.71
11642.38
2879.89
87.94
9.47E−04

C33B95
14774.34
9594.12
1637.99
80.81
6.98E−03

C33B61
13552.71
8211.09
1595.90
106.84
1.83E−03

C33B89
47301.14
34193.78
23334.20
112.80
4.65E−05

C33B122
19740.29
13907.32
5838.25
82.53
1.45E−03

C33B62
12737.71
5620.17
1922.97
934.44
1.32E−03

C33B123
10665.93
10404.03
3232.18
61.08
2.74E−03

C33B54
50466.68
43011.75
38091.89
28785.80
1.35E−04

The panel of mAbs was then further characterized for full affinity analysis as well as epitope binning. The binding of anti-CD33 mAbs to recombinant human CD33 ECD-HSA (C33W2) and cyno CD33 ECD-HSA (C33W1) was measured by ProteOn SPR (Bio-Rad). Goat anti-human Fc IgG (Jackson Immunoresearch, Cat #109-005-098) was directly immobilized via amine coupling at 30 μg/mL in acetate buffer, pH 5.0 on all 6 ligand channels in vertical orientation on a GLC Sensor Chip (Bio-Rad, catalog no. 176-5011) with a flow rate of 30 μL/min in PBS containing 0.005% Tween-20. The immobilization densities averaged about 5000 Response Units (RU) with less than 5% variation among different channels. Different mAbs were captured on the anti-human Fc IgG surface at 0.25 or 0.5 μg/ml (160-300 RU) in vertical ligand orientation, with the 6^thligand channel as no ligand surface control. Human and cyno CD33-HSA proteins at 0.3 μM concentration in 3-fold dilution series of 5 concentrations flew in as analyte to bind to captured mAbs in the horizontal orientation. A buffer sample was also injected in the 6^thchannel to monitor the dissociation of captured mAb and baseline stability. The dissociation phase for all concentrations of human and cyno CD33-HSA was monitored at a flow rate of 100 μL/min for 15 minutes for binding to C33B782, 60 minutes for binding to C33B912 (identical to C33B904 with hIgG4), followed by regeneration using an 18 second pulse of 0.85% phosphoric acid to remove the antigen and the bound mAb. The raw biding data were processed by double referencing after subtracting the response data from: 1) the inter-spot to correct for the non-specific interactions between the Ag and the empty chip surface; 2) the buffer channel to correct for baseline drifting due to the dissociation of captured mAb surface over time. The processed data at all antigen concentrations for each mAb were globally fit to a 1:1 simple Langmuir binding model to extract estimates of the kinetic (k_on, k_off) and affinity (K_d) constants.

To determine whether the panel of mAbs all bind 1 distinct epitope or if there was broad epitope coverage, an epitope binning experiment was performed. Competitive epitope binning of CD33 mAbs was performed on an IBIS SPRi instrument (Carterra) using a CMD-200M sensor prism chip. Each anti-CD33 antibody was directly immobilized via amine coupling on the chip at 10 μg/ml in acetate buffer (pH 4.5) using a separate Continuous Flow Microspotter (CFM). Printed sensor chip was then transferred to the IBIS instrument for the binning analyses using a Classical or “Sandwich” binning format. Binning was performed by sequential injection of human CD33 ECD-HSA, (C33W2) at 50 nM followed by a single anti-CD33 mAb injection as competing analyte in solution at 133 nM to bind immobilized anti-CD33 mAbs with surface regeneration after each sequential injection cycle of antigen and antibody.

To monitor the activity of the immobilized mAbs before and after regeneration, a buffer injection without any competing mAb was performed at the beginning and at the end of the experiment to measure the antigen alone binding activity. The response of competing mAb binding relative to the buffer (antigen alone) binding is an indication whether the antibody in solution blocks or sandwiches the antigen binding to the immobilized mAbs. The raw binning data (.trix file format) were referenced and zeroed using SprintX software (Wasatch, Ver 1.9.3.2), then exported (.ibmx file format) to the binning software HtTools.exe (Wasatch, version 2.0.0.33) for analyses. Data were curated by removing antibodies with antigen responses below RU, and antibodies that did not self-block. Competing mAb responses were normalized relative to the antigen alone binding response. Antibodies with normalized responses <0.25 were denoted blockers, those with normalized responses >0.25 were denoted as non-blockers/Sandwichers. Different bins were predicted using a cut at height 2.5 on the combined dendrogram plot.

The table below summarizes the full kinetic analysis and epitope binning of 32 select mAbs. There are a total of 8 anti-CD33 mAbs that have sub-nanomolar affinity for both human and cyno CD33 and these mAbs correspond to 3 distinct epitope bins while the larger panel has a range of affinities and 7 distinct epitope bins.

TABLE 47

Full Kinetics Analysis and Epitope Binning of OMNIRat derived mAbs

Human CD33 ECD-HSA

Epitope

Prot. AA ID
V Region ID
ka (1/Ms)
k_d(1/s)
K_D(M)
Bin

C33B48
C33F53
1.62E+06
1.82E−05
1.12E−11
1

C33B46
C33F51
1.45E+06
1.99E−03
1.38E−09
1

C33B66
C33F71
3.85E+04
2.03E−03
5.29E−08
1

C33B107
C33F112
binding/no fit
binding/no fit
binding/no fit
1

C33B88
C33F93
binding/no fit
binding/no fit
binding/no fit
1

C33B96
C33F101
2.26E+05
4.36E−04
1.92E−09
3

C33B101
C33F106
1.62E+05
1.08E−03
6.64E−09
3

C33B73
C33F78
5.59E+05
5.59E−05
1.00E−10
4

C33B125
C33F130
9.92E+05
1.34E−04
1.40E−10
4

C33B55
C33F60
9.85E+05
2.53E−04
2.60E−10
4

C33B82
C33F87
4.45E+05
2.70E−04
6.10E−10
4

C33B83
C33F88
2.70E+05
5.21E−04
1.93E−09
4

C33B75
C33F80
3.85E+05
4.41E−03
1.14E−08
4

C33B123
C33F128
1.02E+06
1.52E−02
1.48E−08
4

C33B52
C33F57
2.06E+05
3.96E−03
1.92E−08
4

C33B61
C33F66
4.89E+05
1.05E−02
2.14E−08
4

C33B62
C33F67
5.07E+05
1.26E−02
2.49E−08
4

C33B64
C33F69
4.33E+05
2.21E−03
5.10E−09
4

C33B63
C33F68
5.33E+05
3.74E−03
7.01E−09
4

C33B122
C33F127
7.47E+05
7.12E−03
9.53E−09
4

C33B72
C33F77
8.71E+05
2.00E−04
2.30E−10
5

C33B79
C33F84
5.15E+05
3.90E−04
7.60E−10
5

C33B77
C33F82
8.28E+05
2.62E−04
3.20E−10
6

C33B87
C33F92
7.20E+05
4.32E−04
6.00E−10
6

C33B94
C33F99
9.22E+05
5.85E−04
6.30E−10
6

C33B95
C33F100
4.82E+05
7.40E−03
1.54E−08
6

C33B120
C33F125
5.75E+05
1.68E−03
2.93E−09
6

C33B89
C33F94
low binding
low binding
low binding
8

C33B54
C33F59
low binding
low binding
low binding
9

C33B124
C33F129
3.57E+05
1.24E−04
3.50E−10
NB

C33B80
C33F85
3.23E+05
4.25E−04
1.32E−09
NB

C33B56
C33F61
low binding
low binding
low binding
NB

Cyno CD33 ECD-HSA

Epitope

Prot. AA ID
V Region ID
ka (1/Ms)
k_d(1/s)
K_D(M)
Bin

C33B48
C33F53
4.31E+06
1.58E−04
3.66E−11
1

C33B46
C33F51
2.97E+06
3.75E−04
1.26E−10
1

C33B66
C33F71
1.22E+06
2.66E−04
2.17E−10
1

C33B107
C33F112
3.31E+05
7.01E−05
2.12E−10
1

C33B88
C33F93
binding/no fit
binding/no fit
binding/no fit
1

C33B96
C33F101
binding/no fit
binding/no fit
binding/no fit
3

C33B101
C33F106
2.25E+05
2.69E−04
1.20E−09
3

C33B73
C33F78
6.00E+05
5.08E−04
8.46E−10
4

C33B125
C33F130
1.12E+06
3.39E−04
3.04E−10
4

C33B55
C33F60
1.16E+06
8.37E−05
7.23E−11
4

C33B82
C33F87
5.45E+05
7.51E−04
1.38E−09
4

C33B83
C33F88
2.47E+05
2.88E−03
1.17E−08
4

C33B75
C33F80
6.16E+05
1.32E−03
2.15E−09
4

C33B123
C33F128
1.26E+06
3.39E−03
2.69E−09
4

C33B52
C33F57
3.13E+05
1.48E−03
4.74E−09
4

C33B61
C33F66
7.34E+05
1.62E−03
2.21E−09
4

C33B62
C33F67
8.05E+05
1.49E−03
1.85E−09
4

C33B64
C33F69
5.90E+05
1.01E−03
1.71E−09
4

C33B63
C33F68
7.23E+05
8.80E−04
1.22E−09
4

C33B122
C33F127
binding/no fit
binding/no fit
binding/no fit
4

C33B72
C33F77
9.19E+05
5.40E−04
5.87E−10
5

C33B79
C33F84
5.48E+05
2.20E−03
4.01E−09
5

C33B77
C33F82
1.08E+06
2.66E−04
2.47E−10
6

C33B87
C33F92
1.12E+06
2.64E−04
2.36E−10
6

C33B94
C33F99
1.10E+06
5.20E−04
4.73E−10
6

C33B95
C33F100
8.44E+05
8.06E−03
9.56E−09
6

C33B120
C33F125
8.76E+05
9.02E−04
1.03E−09
6

C33B89
C33F94
2.65E+05
2.01E−04
7.60E−10
8

C33B54
C33F59
1.32E+06
6.37E−04
4.84E−10
9

C33B124
C33F129
4.67E+05
4.72E−04
1.01E−09
NB

C33B80
C33F85
4.92E+05
2.59E−04
5.27E−10
NB

C33B56
C33F61
low binding
low binding
low binding
NB

The OmniMouse panel (8 mAbs total) was generated separately and characterized further for binding to cells. Cell binding was performed as described above and summarized in the table below. Of the 8 mAbs tested 6 bound directly to CD33 expressing cells while 2 did not.

TABLE 48

Cell Binding of OMNIMouse derived mAbs to human and cyno expressing cell lines

Parental
Human CD33

mAb
60 nM
6 nM
0.6 nM
0 nM
60 nM
6 nM
0.6 nM
0 nM

C33B909
253.50
206.04
169.77
119.51
176.49
170.25
154.00
191.28

C33B910
193.52
176.14
108.46
190.17
213.55
183.33
151.25
155.29

C33B911
1466.02
389.41
186.22
113.30
237954.27
100333.48
13501.02
114.07

C33B912
977.91
273.07
140.62
124.53
237140.86
101295.70
15726.96
149.54

C33B913
174.49
118.08
123.26
129.07
518952.00
409071.06
204694.14
127.82

C33B914
181.37
142.74
139.10
113.48
304350.88
315129.56
153252.58
185.45

C33B915
101.28
147.65
143.51
100.00
390477.25
362902.66
138398.56
112.22

C33B916
416.08
145.16
115.70
91.75
447815.47
404033.19
192941.55
167.07

Cyno CD33

mAb
60 nM
6 nM
0.6 nM
0 nM

C33B909
180.33
135.33
115.73
124.03

C33B910
202.42
135.18
116.71
175.97

C33B911
17036.56
7729.14
1935.16
97.94

C33B912
15070.88
7271.38
1726.03
124.69

C33B913
40661.90
36920.95
35224.10
106.19

C33B914
44964.85
33368.26
22086.01
86.76

C33B915
37495.34
35692.21
36165.59
113.92

C33B916
41004.43
33294.78
22790.61
104.43

The 6 mAbs that bound CD33 on cells were further characterized biophysically via full kinetic analysis to recombinant antigen using the methods described above and summarized in the table below. Of the 6 mAbs tested, 1 bound to human CD33 with a picomolar affinity (C33B912) and subnamolar for cyno CD33, while 1 had very strong affinity for human CD33 but only nanomolar affinity towards cyno CD33 (C33B911). Two more clones were subnanomolar for both human and cyno CD33 (C33B913 and C33B916), but neither affinity was 15 in the range of C33B912.

TABLE 49

Full Kinetics Analysis of OMNIMouse derived mAbs

Human CD33 ECD-HSA
Cyno CD33 ECD-HSA

mAb
k_a(1/Ms)
k_d(1/s)
K_D(M)
k_a(1/Ms)
k_d(1/s)
K_D(M)

C33B911
1.10E+06
4.14E−05
3.78E−11
1.15E+06
1.15E−03
1.00E−09

C33B912
1.42E+06
4.29E−05
3.02E−11
1.50E+06
6.50E−04
4.33E−10

C33B913
6.60E+05
6.40E−04
9.69E−10
2.56E+06
3.08E−04
1.20E−10

C33B914
4.44E+05
9.80E−03
2.21E−08
5.29E+05
2.33E−04
4.40E−10

C33B915
2.18E+05
9.89E−04
4.53E−09
3.81E+06
8.93E−05
2.34E−11

C33B916
6.27E+05
4.11E−04
6.55E−10
4.73E+05
4.03E−04
8.52E−10

An epitope binning experiment was performed on the 6 cell binding mAbs derived from OMNIMouse along with several control mAbs previously identified in the earlier OMNIRat campaign. The control mAbs were chosen based on their subnanomolar affinity towards human CD33 and the number of distinct epitope bins. The binning software HtTools assigns Epitope Bin numbers on a per experiment basis and therefore having several controls to already defined epitope bins was critical for cross-comparison. The two OMNIMouse derived human CD33 high affinity clones (C33B911 and C33B912) both binned with clones from bin 4 above (bin 4 in this experiment) while the subnanomolar clone (C33B916) binned into 2 here along with C33B836 (bin 1 in the above experiment).

TABLE 50

Epitope Bins of OMNIMouse anti-CD33 mAbs

mAb
V region ID
Epitope Bin

C33B915
C33F553
1

C33B916
C33F554
2

C33B836
C33F53
2

C33B914
C33F552
2

C33B913
C33F551
3

C33B806
C33F106
3

C33B911
C33F549
4

C33B912
C33F550
4

C33B778
C33F78
4

C33B830
C33F130
4

C33B782
C33F82
5

C33B792
C33F92
5

C33B799
C33F99
5

C33B760
C33F60
6

C33B777
C33F77
7

CD33 is comprised of 2 IgG domains, the membrane distal V domain and the membrane proximal C2 domain. The SNP rs12459419 can cause the selective alternative splicing of the CD33 pre-mRNA transcript to yield a C2 only form expressed on cells and therefore targeting this domain can provide clinical benefit. To ascertain which of the two domains, the mAbs were capable of binding, an off-rate screen was performed following the protocol above on 6 mAbs with the highest binding capability that covered 4 distinct epitope bins using Human CD33 ECD-HSA, Human CD33 V-HSA and Human CD33 C2-HSA as the binding antigens. As shown in the table below, the two clones previously grouped in bin 4 both bound to the huCD33 C2 domain but not the huCD33 V domain, while the clones in bin 2 and 3 bound the V domain but not the C2 domain. Two clones grouped into bin 5 did not bind either domain, and, therefore, their exact binding location could span the two domains. Three (3) commercially available mAbs were included in this experiment ((WM53 (EMD Millipore; Darmstadt, Germany), P67.7 (Biolegend, San Diego, CA), and LSBio clone 906 (LifeSpan Biosciences, Seattle, WA))) and all showed binding to the V domain, but not the C2 domain. Looking at the epitope bins in Tables 42 and 45 in relation to the C2 domain binding data in Table 51, there are a total of 15 mAbs that could potentially bind the C2 domain ranging in affinities from ˜25 nM to ˜30 pM on the human full length protein.

TABLE 51

Off-rate Domain Binding

huCD33 ECD-
huCD33-V-
huCD33-C2-

Protein
HSA
HSA
HSA
Epitope

ID
k_d(1/s)
k_d(1/s)
k_d(1/s)
Bin

C33B912
1.29E−05
No/low binding
6.68E−05
4

response

C33B778
4.72E−05
No/low binding
2.57E−03
4

response

C33B782
2.58E−04
No/low binding
No/low binding
5

response
response

C33B792
4.27E−04
No/low binding
No/low binding
5

response
response

C33B836
5.52E−05
3.71E−05
No/low binding
2

response

C33B806
1.36E−03
3.18E−03
No/low binding
3

response

WM53
2.37E−03
3.78E−02
No/low binding

response

P67.7
1.05E−03
2.43E−03
No/low binding

response

LSBio
2.45E−03
4.34E−02
No/low binding

clone 906

response

To support further in vivo and in vitro studies, select clones (C33B836, C33B782, C33B778, C33B904, C33B806, C33B830, C33B937, C33B792, C33B760, and C33B777) were chosen for scale-up and fab arm exchange to produce bi-specific molecules with anti-CD3 antibodies. ExpiCHO-S™ cells (ThermoFisher Scientific) were seeded at 1.25×10⁵-2.25×10⁵viable cells/mL in ExpiCHO™ Expression Medium and cultured in polycarbonate, disposable, sterile, vented, non-baffled Erlenmeyer shake flasks in a 37° C., 7% CO2 shaker incubator (INFORS HT Multitron Pro). For routine cell growth in 125 mL-2 L shake flasks, the shake speed was set to 130 rpm for shakers with a 19 mm shaking diameter. Cells were sub-cultured when the density reached log phase growth at 4×10⁶-6×10⁶viable cells/mL with a 98-99% viability.

Two days before transfection, ExpiCHO-S™ cells were seeded at 1.5×10⁶viable cells/mL for the required culture volume. On the day of transfection, the viable cell density and percent viability was determined. Cells were transfected at a density of 6×10⁶viable cells/mL. For optimal transfection, sterile heavy and light chain plasmid DNA at >1 mg/mL concentration in TE buffer (10 mM Tris-HC1, 1 mM EDTA, pH 8.0) was used.

ExpiCHO-S™ cells were transfected following manufacturer's Max Titer Transfection protocol (ThermoFisher Publication Number MAN0014337). All amounts and volumes shown below were per mL of the final transfected culture volume. Briefly, plasmid DNA was diluted with 0.04 mL cold OptiPRO™ medium (ThermoFisher Scientific) at the following ratio: 0.125 μg Heavy Chain DNA: 0.375 μg Light Chain DNA: 0.5 μg pAdvantage. 6.4 μL of ExpiFectamine™ CHO Transfection Reagent was diluted and mixed gently with 0.04 mL cold OptiPRO™ medium and incubated for 1 min. The diluted ExpiFectamine™ CHO Reagent was added to the diluted DNA, mixed gently and the ExpiFectamine™ CHO/plasmid DNA complexes were incubated at room temperature for 5 minutes. Post-incubation, the complexes were added to the ExpiCHO-S™ cells in a shaker flask and incubated overnight in a 37° C., 7% CO₂shaker incubator.

For the Max Titer protocol, on Day 1 post-transfection, 6 μL ExpiFectamine™ CHO Enhancer and 1604, ExpiCHO™ Feed were added and the flask was transferred to a 32° C., 7% CO2 shaker incubator. On Day 5 post-transfection, 1604, of ExpiCHO™ Feed was added for the second time to the flask and returned to the 32° C. incubator with shaking. The culture was harvested on Day 12 post-transfection, centrifuged at 5000 rpm for 15 mins and clarified through a 0.2 um Acropak 1500 filter capsule (Pall).

Expressed antibodies were purified from the clarified supernatants using MabSelect SuRe (GE Healthcare). MabSelect SuRe Protein A columns were equilibrated with 1×D-PBS, pH 7.2 prior to loading individual culture supernatants. Unbound proteins were removed by washing extensively with 1×D-PBS, pH 7.2. Bound proteins were eluted with 0.1 M Na-acetate, pH 3.5. Peak fractions were neutralized with 2.5M Tris pH 7.2 and pooled. The neutralized fraction pools were either dialyzed into 1×dPBS for assays and biophysical characterization or utilized for bispecific assembly.

The protein concentration for each elution pool was determined by measuring absorbance at OD280 nm and calculated using absorbance extinction coefficient based on the amino acid sequence.

6 Preparation and Functional Evaluation of CD33×CD3 Bispecific Antibodies
6-1 Fab-Arm Exchange Using Purified Parental mAbs

The formation of the CD33×CD3 bispecific antibodies requires two parental mAbs, one specific for the targeting arm (e.g. CD33) and one specific for the effector arm (e.g. CD3). CD33 mAbs were recombined with a high affinity (CD3B376: VH of SEQ ID NO: 652, and VL of SEQ IF NO: 661) or low affinity CD3 arm (CD3B450: VH of SEQ ID NO: 657, and VL of SEQ IF NO: 678) arms. These parental mAbs (CD33 and CD3 arms) are in the IgG4 PAA format (Labrijn et al, 2013) where the targeting parent (CD33) contains the 409R Genmab mutation (native amino acid for IgG4), while the killing parent (CD3) contains the F405L mutation and R409K. The monospecific anti-CD3 antibody was expressed as IgG4, having Fc substitutions S228P, F234A, L235A, F405L, and R409K (CD3 arm) (numbering according to EU index) in their Fc regions. The monospecific antibodies were expressed and purified as described above. Post purification the parental CD33 antibodies were mixed with the desired parental CD3 antibody under reducing conditions in 75 mM 2-MEA (2-Mercaptoethylamine) and incubated at 31° C. for 5 hours or at room temperature overnight. The recombination reactions were based on molar ratios, where a set amount of CD33 antibody (e.g., 10 mg, or ˜74.6 nanomoles) was combined with CD3 antibody (e.g., −67.8 nanomoles), where the CD33 antibody was added in a 6% excess of the CD3 antibody. The concentrations of the CD33 antibody stocks varied from 0.8 to 6 mg/mL, and the volumes of the recombination reactions varied for each pairing. The recombination reactions were subsequently dialyzed overnight against PBS to remove the reductant. The CD33×CD3 bispecific antibody reactions were performed with an excess of the CD33 antibody (ratio) to minimize the amount of unreacted CD3 parental antibody remaining after recombination.

The final CD33×CD3 bispecific antibodies produced, along with the parental mAbs (i.e. CD33, CD3, or Null) used in the recombination reactions are listed in Table 47.

Selected CD33 hits were also paired with a non-killing arm (Null) to create negative controls for testing purposes. For control bispecific antibodies, B2M1, an RSV antibody in the IgG4 PAA format was generated, purified and, combined with either the CD3 arms CD3B219 or CD3B376-F405L, R409K to generate CD3B288 (CD3×Null) and CD3B510 (CD3B376×Null), respectively; CD33 arms were combined with B23B49 to generate CD33×Null, as in Table 52.

TABLE 52

CD33 × CD3 bispecific antibodies

VH Pep

VL Pep

Bispecific Ab
Parental
HC Pep ID
SEQ ID
LC Pep ID
SEQ ID

C3CB7
C33B836
C33H80
267
C33L73
306

CD3B219
CD3H141
247
CD3L66
287

C3CB5
C33B830
C33H84
269
C33L66
308

CD3B219
CD3H141
247
CD3L66
287

C3CB4
C33B806
C33H69
260
C4LL152
300

CD3B219
CD3H141
247
CD3L66
287

C3CB16
C33B799
C33H98
275
C33L69
314

CD3B219
CD3H141
247
CD3L66
287

C3CB14
C33B792
C33H87
270
C33L35
309

CD3B219
CD3H141
247
CD3L66
287

C3CB12
C33B782
C33H72
262
C33L40
301

CD3B219
CD3H141
247
CD3L66
287

C3CB11
C33B778
C33H66
258
C33L60
298

CD3B219
CD3H141
247
CD3L66
287

C3CB10
C33B777
C33H65
257
C33L47
297

CD3B219
CD3H141
247
CD3L66
287

C3CB8
C33B760
C33H45
250
C33L11
290

CD3B219
CD3H141
247
CD3L66
287

C3CB97
C33B836
C33H80
267
C33L73
306

CD3B376
CD3H219
652
CD3L150
661

C3CB98
C33B830
C33H84
269
C33L66
287

CD3B376
CD3H219
652
CD3L150
661

C3CB99
C33B806
C33H69
260
C4LL152
300

CD3B376
CD3H219
652
CD3L150
661

C3CB100
C33B799
C33H98
275
C33L69
314

CD3B376
CD3H219
652
CD3L150
661

C3CB101
C33B792
C33H87
270
C33L35
309

CD3B376
CD3H219
652
CD3L150
661

C3CB102
C33B782
C33H72
262
C33L40
301

CD3B376
CD3H219
652
CD3L150
661

C3CB103
C33B778
C33H66
258
C33L60
298

CD3B376
CD3H219
652
CD3L150
661

C3CB104
C33B777
C33H65
257
C33L47
297

CD3B376
CD3H219
652
CD3L150
661

C3CB105
C33B760
C33H45
250
C33L11
290

CD3B376
CD3H219
652
CD3L150
661

C33B941
C33B836
C33H80
267
C33L73
306

B23B49
B23H1
246
B23L3
286

C33B942
C33B830
C33H84
269
C33L66
308

B23B49
B23H1
246
B23L3
286

C33B943
C33B806
C33H69
260
C4LL152
300

B23B49
B23H1
246
B23L3
286

C33B944
C33B799
C33H98
275
C33L69
314

B23B49
B23H1
246
B23L3
286

C33B945
C33B792
C33H87
270
C33L35
309

B23B49
B23H1
246
B23L3
286

C33B946
C33B782
C33H72
262
C33L40
301

B23B49
B23H1
246
B23L3
286

C33B947
C33B778
C33H66
258
C33L60
298

B23B49
B23H1
246
B23L3
286

C33B948
C33B777
C33H65
256
C33L47
297

B23B49
B23H1
246
B23L3
286

C33B949
C33B760
C33H45
250
C33L11
290

B23B49
B23H1
246
B23L3
286

CD3B288
B23B39
B23H1
246
B23L3
286

CD3B219
CD3H141
247
CD3L66
287

CD3B510
B23B39
B23H1
246
B23L3
286

CD3B376
CD3H219
652
CD3L150
661

C3CB87
C33B903
C33H251
280
C33L117
319

CD3B219
CD3H141
247
CD3L66
287

C3CB88
C33B904
C33H252
281
C33L118
320

CD3B219
CD3H141
247
CD3L66
287

C3CB89
C33B905
C33H253
282
C33L119
321

CD3B219
CD3H141
247
CD3L66
287

C3CB90
C33B907
C33H255
284
C33L121
323

CD3B219
CD3H141
247
CD3L66
287

C3CB91
C33B908
C33H256
285
C33L122
324

CD3B219
CD3H141
247
CD3L66
287

C3CB189
C33B904
C33H252
281
C33L118
320

CD3B376
CD3H219
652
CD3L150
661

Pep: Peptide;

Nuc: Nucleotide;

SEQ ID: SEQ ID NO

6-2 Ex Vivo CD33×CD3 Mediated Reduction of AML Blasts and T Cell Activation in an AML Primary Sample

To further assess the cytotoxicity potential of CD33×CD3 bispecific antibodies, an ex vivo cytotoxicity assay was performed using AML, patient whole blood using the top four antibodies (FIG. 61). In this assay, various bispecific antibodies (CD33 antibodies paired with either CD3 arm CD3B219 and CD3B376) were added to diluted whole blood from AML patients for a period of 48 hours without providing additional T-cells, since this assay relies on the presence of autologous T-cells in the patient's blood. At 48 hours, the samples were stained with CD3 PerCPCy5.5, CD25 PE, CD33 FITC and CD38 APC (all antibodies were purchased from Biolegend; San Diego, CA). The samples were then washed at least 3 times in 1× Lyse RBC Lysis Buffer (eBioscience). The samples were then stained with the LIVE/DEAD® Fixable Near-IR Dead Cell Stain buffer (Life Technologies). The extent of tumor cytotoxicity was determined by first quantifying the live CD33^{+ cells in the fraction of AML patient cancer cells (defined as CD}3⁻CD38⁺ cells) in the presence of the bispecific antibodies. Cytotoxicity was calculated as a percentage relative to PBS/untreated control using the following equation: (% CD33^{+ in PBS/untreated control-% CD}33^{+ in treated sample)/(% CD}33^{+ in PBS/untreated control). T cell activation was calculated as a percentage of CD}25⁺ events in CD3⁺ fraction.

As shown in FIG. 61, all CD33 lead antibodies paired with either CD3 arm (CD3B376 and CD3B219) promoted a dose-dependent reduction of total cytotoxicity that correlated with T cell activation after 48 hours. Null arm control antibodies (Null×CD3B219 and null×CD3B376) failed to show tumor cell cytotoxicity or T cell activation. This result also demonstrated that the CD33×CD3 bispecific antibodies work in an autologous setting. These results are representative of 4 other AML donor samples (data not shown). Table 53 summarizes the EC₅₀values generated with the CD33×CD3 multispecific antibodies. As seen from the EC₅₀values, C33B904 paired with either CD3 arm (C3CB88, C3CB189) as well C33B836 paired with either CD3 arm (C3CB7, C3CB97) were the most potent and efficacious antibodies. These 4 antibodies were thus the focus of further characterization.

TABLE 53

CD33 × CD3 T-cell mediated ex vivo cytotoxicity

assays. Summary of the EC₅₀values for 8

CD33 × CD3 bispecific antibodies

Bispecifc Ab
Primary AML Cell

ID
Killing EC₅₀(nM)

C3CB11
3.958

C3CB12
2.635

C3CB7
0.3315

C3CB88
0.6722

C3CB103
4.186

C3CB102
4.973

C3CB97
~0.2316

C3CB189
0.5782

6-3 Demonstration that the CD33×CD3 Bispecific Antibodies Bind to the C2 Domains of CD33 and Induce Cytotoxicity of CD33 Single Nucleotide Polymorphism (SNP) Expressing Cell Lines
In Vitro T Cell Mediated Cytotoxicity Assays with CD33×CD3 Bispecific Antibodies

Recent studies showed that a single nucleotide polymorphism (SNP) rs12459419 was present in ˜50% of the AML population and leads to skipping of exon 2 of CD33 which results in the deletion of the V domain of CD33. This study also showed that Mylotarg which binds to the V domain of CD33, had no efficacy in patients that express the SNP, and, therefore, reduced risk of relapse and improved survival in ˜50% of the AML population (Lamba et al 2017, JCO, CD33 Splicing Polymorphism Determines Gemtuzumab Ozogamicin Response in De Novo Acute Myeloid Leukemia: Report From Randomized Phase III Children's Oncology Group Trial AAML0531). Given the data with Mylotarg in the above mentioned study, in vitro T cell mediated cytotoxicity assays were performed to assess whether CD33 hits (V binding C33B836 vs C2 binding C33B904) paired with CD3 arms (CD3B219 or CD3B376) mediate killing of SNP rs12459419 expressing cell lines. Briefly, effector cells (pan T cells purchased from Biological Specialty) were harvested, counted, washed, and resuspended to 1×10⁶cells/ml in RPMI (Invitrogen) with 10% FBS (Invitrogen) cell media. Target cells (KG1, SH2 and OCIAML3) were labeled with CFSE (Invitrogen) and resuspended to 2×10⁵cells/mL in RPMI with 10% FBS. KG1, SH2 and OCIAML3 were chosen to represent wildtype, heterozygous and homozygous for the CD33 SNP rs12459419 mutation, respectively. Effectors and CFSE-labeled target cells were mixed at effector:target ratio (E:T)=5:1 in sterile 96-well round bottom plates. 10 μl of Fc block (ReoPro Fc fragment) along with 5 μL aliquot of bispecific antibody was added to each well containing various concentrations. Cultures were incubated at 37° C. for 48 hours under 5% CO2. After 48 hrs, the LIVE/DEAD® Fixable Near-IR Dead Cell Stain buffer (Life Technologies) was added to samples and cultures were incubated for 20 min in the dark at RT, washed, and resuspended in 100-200 μL FACs buffer. The drug-induced cytotoxicity was determined using CANTO II flow cytometer (BD Biosciences) and analyzed with FlowJo Software or Dive software (BD Biosciences). The population of interest is the double positive CFSE+/live/dead+ cells. As shown in FIG. 62, unlike the null arm controls (null×CD3B219 and null×CD3B376), V binding and C2 binding CD33×CD3 multispecific antibodies induced T cell redirected cell cytotoxicity of CD33+WT for SNP rs12459419 mutation cell line KG1 at 48 hrs. In contrast, unlike V binder C33B836 (C3CB97, C3CB7), only the C2 binding C33B904 paired bispecific antibodies (C3CB189, C3CB88) mediated cytotoxicity of SH2 and OCIAML3 cell lines that were heterozygous or homozygous for the rs12459419 SNP mutations, respectively. For this reason, C33B904 paired bispecific antibodies (C3CB189, C3CB88) were taken forward for further analysis and characterization. Collectively, these data suggest that CD33 C2 binding bispecific antibodies such as C33B904 paired bispecific antibodies have the potential to show efficacy in a broader group of AML patients than V binding competitor anti-CD33 antibodies.

6-4 Ex Vivo CD33×CD3 Mediated Reduction of Spiked in MOLM-13 and Monocytes in an Ex Vivo Whole Blood MOLM-13 Cytotoxicity Assay

To assess the cytotoxicity potential of CD33×CD3 bispecific antibodies at eliminating spiked in MOLM-13 cells and normal human monocytes, an ex vivo cytotoxicity assay using normal healthy human whole blood with exogenously added CD33^{+ AML cell line MOLM-}13 was utilized. Similar to the above experiment, various bispecific antibodies (CD33 antibodies paired with either CD3 arm CD3B219 and CD3B376) were added to diluted whole blood from 6 different normal human donors for a period of 48 hr without providing additional T-cells, since this assay relies on the presence of autologous T-cells in the donor's blood. Prior to dilution, the concentration of T cells in the blood of each donor was enumerated. The blood was then diluted with CFSE (Invitrogen) labeled MOLM-13 cells, such that effector:target ratio (E:T) is 1:5 to mimic the effector: target ratio in AML patient samples. At 48 hs, the samples were stained with CD3 PerCPCy5.5, CD25 PE, CD33 FITC and CD14 Pacific Blue (all antibodies were purchased from Biolegend). The samples were then washed at least 3 times in 1× Lyse RBC Lysis Buffer (eBioscience). The samples were then stained with the LIVE/DEAD® Fixable Near-IR Dead Cell Stain buffer (Life Technologies). The extent of tumor cytotoxicity was determined by first quantifying the live CD33^{+ cells in the fraction of CD}14⁺ monocytes in the presence of the bispecific antibodies. Cytotoxicity of MOLM-13 cells was determined by enumerating the percentage of dead CFSE⁺ cells. Cytotoxicity of monocytes was calculated as a percentage relative to PBS/untreated control using the following equation: (% CD33⁺CD14⁺ in PBS/untreated control-% CD33⁺CD14⁺ in treated sample)/(% CD33⁺CD14⁺ in PBS/untreated control). The data in FIG. 63 indicate that both CD33×CD3 bispecific antibodies (same CD33 lead C33B904 paired with either CD3 arm, CD3B376 and CD3B219) specifically induce cell cytotoxicity of MOLM-13 cells and CD33^{+ monocytes at}48 hr. The null arm controls were used as negative bispecific antibody controls. The null arm control showed little-to-no cytotoxicity activity of the MOLM-13 and CD33^{+ monocytes. These data show the average values of}6 different normal donors. The average EC₅₀values for cytotoxicity of MOLM-13 and CD14⁺ monocytes are shown in Table 54.

TABLE 54

CD33 × CD3 T-cell mediated ex vivo cytotoxicity assays. Summary

of EC₅₀values for 2 CD33 × CD3 bispecific antibodies.

CD33⁺ CD14⁺

Bispecific Ab
MOLM13 Killing EC₅₀
Killing EC₅₀

ID
(nM)
(nM)

C3CB189
0.1677
1.156

C3CB88
0.671
0.506

6-5 Demonstration of Species Cross-Reactivity of CD33×CD3 Bispecific Antibodies to Cynomologous Monkey
Ex Vivo CD33×CD3 Mediated Reduction of Monocytes in an Ex Vivo Cytotoxicity Assay with Cynomologous Whole Blood

To demonstrate functional cross-reactivity and to assess the cytotoxicity potential of CD33×CD3 bispecific antibodies at eliminating normal cynomologous monocytes, an ex vivo cytotoxicity assay using healthy cynomologous whole blood was utilized. Similar to the above experiment, various bispecific antibodies (CD33 antibodies paired with either CD3 arm CD3B219 and CD3B376) were added to diluted whole blood from 6 different normal cynomologous monkey donors for a period of 48 hr without providing additional T-cells, since this assay relies on the presence of autologous T-cells in the donor's blood. At 48 hrs, the samples were stained with CD3 PerCPCy5.5, CD25 PE, CD33 FITC and CD14 Pacific Blue (all antibodies were purchased from Biolegend except for the CD33 antibody which was purchased from Miltenyi; Bergisch Gladbach, Germany). The samples were then washed at least 3 times in 1× Lyse RBC Lysis Buffer (eBioscience) prior to staining with the LIVE/DEAD® Fixable Near-IR Dead Cell Stain buffer (Life Technologies). The extent of monocyte cytotoxicity was determined by first quantifying the live CD33^{+ cells in the fraction of CD}14⁺ monocytes in the presence of the bispecific antibodies. Cytotoxicity was calculated as a percentage relative to PBS/untreated control using the following equation: (% CD33⁺CD14⁺ in PBS/untreated control-% CD33⁺CD14⁺ in treated sample)/(% CD33⁺CD14⁺ in PBS/untreated control). T cell activation was calculated as a percentage of CD25⁺ events in CD3⁺ fraction. The data in FIG. 64 indicated that both CD33×CD3 bispecific antibodies (same CD33 lead C33B904 paired with either CD3 arm, CD3B376 and CD3B219) specifically induced cell cytotoxicity of CD33^{+ monocytes as well as T cell activation at}48 hr. The null arm controls were used as negative bispecific antibody controls and showed little-to-no cytotoxicity or T cell activity. Table 55 shows the average values of 6 different cynomologous donors.

TABLE 55

CD33 × CD3 T-cell mediated ex vivo cytotoxicity assays. Summary

of the EC₅₀values for 2 CD33 × CD3 bispecific antibodies.

CD33⁺ CD14⁺
T cell

Protein AA ID
Killing EC₅₀(nM)
activation EC₅₀(nM)

C3CB189
3.60
0.02

C3CB88
0.89
0.02

6-6 Efficacy of C3CB189 and C3CB88 in MOLM-13 Human AML Xenografts in T Cell Humanized NSG Mice

Efficacy of C3CB189 and C3CB88 was evaluated in established luciferase-transfected disseminated MOLM-13 human acute myeloid leukemia (AML) xenografts in female NOD.Cg-Prkdc^scidIl2rg^tm1Wjl/SzJ (NSG) mice humanized with 20 million T cells. Animals were randomized into n=10/group by live bioluminescence imaging (BLI) on day 5 post-i.v. tumor implantation. C3CB189 and C3CB88 at 0.005, 0.05 and 0.5 mg/kg or Null×CD3 antibody control at 0.5 mg/kg were dosed i.p. every 3-4 days for 6 weeks.

On day 13 post-tumor implantation, when at least eight animals remained per group, tumor growth inhibition (% TGI) as determined by bioluminescence was calculated. Statistically significant tumor growth inhibition was observed with C3CB189 (FIG. 65) and C3CB88 (FIG. 67) at all concentrations, as compared to Null×CD3 control. C3CB189 at 0.005, 0.05, and 0.5 mg/kg elicited tumor growth inhibition of 76%, 100% and 82%, respectively, and C3CB88 at 0.005, 0.05, and 0.5 mg/kg elicited tumor growth inhibition of 100%, 100% and 91%, respectively, as compared to Null×CD3 treated controls.

Treatment with C3CB189 and C3CB88 resulted in reduced tumor burden and increased life span (ILS) greater than the 16-day median survival of the Null×CD3 control group. Animals treated with C3CB189 had a median survival of 19-27.5 days (FIG. 66) and animals treated with C3CB88 had a median survival of 26-28.5 (FIG. 68) days across doses. C3CB189 at 0.005, 0.05, and 0.5 mg/kg resulted in an increased life span of 19%, 72% and 50%, respectively, and C3CB88 resulted in an increased life span of 63%, 78% and 72%, respectively, as compared to the control group.

7 TMEFF2 Antibodies
7-1 Antigen Generation

Human extracellular domain (ECD) TMEFF2 was produced based on UniProt Accession #Q9UIK5 sequence. The ECD construct was designed with a 6× His-tag (SEQ ID NO: 596) and Avi-tag sequences at the C-terminus (construct TMEW1; SEQ ID NO: 578). A construct containing the FS2 and EGF domains (amino acids 151-320 was designed as a human serum albumin (HSA) fusion with a 6×His-tag (SEQ ID NO: 596) and avitag sequences (construct TMEW7; SEQ ID NO: 579). A construct containing the TMEFF2 membrane proximal domain (residues 230-320) was designed with a 6×His tag (SEQ ID NO: 596) (construct TMEW19; SEQ ID NO: 580) or fused to a rat IgG1 Fc with a His-tag (construct TMEW20; SEQ ID NO: 581). Residues 230-320 of TMEFF2 contain the EGF domain which spans the residues 261-301 of TMEFF2. The human TMEFF2 ECD expression constructs were transiently transfected into HEK293 derived cells, Expi293 (Gibco/Thermo Fisher Scientific) using Expifectamine according to manufacturer protocol. Cells were incubated 5 days at 37° C. with 8% CO2 on an orbital shaker before harvesting. The expressed cells were removed by centrifugation and the soluble TMEFF2 proteins with his-tags were purified from the media using immobilized metal affinity chromatography using Ni Sepharose 6 Fast Flow resin (GE Healthcare) followed by Superdex 200 preparative size exclusion chromatography (SEC) (GE Healthcare) in Dubelcco's Phosphate Saline buffer pH 7.2 (1×DPBS). The amino acid sequences of the generated antigens are shown in Table 58.

TABLE 58

Protein AA ID
Description
Amino Acid Sequence

TMEW1
TMEFF2-FL-ECD-His-
FPTSLSDCQTPTGWNCSGYDDRENDLFLCDTNTCK

(SEQ ID NO: 578)
Avitag
FDGECLRIGDTVTCVCQFKCNNDYVPVCGSNGESY

QNECYLRQAACKQQSEILVVSEGSCATDAGSGSGD

GVHEGSGETSQKETSTCDICQFGAEVDEDAEDVWC

VCNIDCSQTNFNPLCASDGKSYDNACQIKEASCQK

QEKEIVMSLGRCQDNTTTTTKSEDGHYARTDYAEN

ANKLEESAREHHIPCPEHYNGFCMHGKCEHSINMQ

EPSCRCDAGYTGQHCEKKDYSVLYVVPGPVRFQYV

GGGSHHHHHHLNDIFEAQKIEWHE

TMEW7
FS2-EGF-Tev-
SGETSQKETSTCDICQFGAEVDEDAEDVWCVCNID

(SEQ ID NO: 579)
HSA(C34S)-His-
CSQTNFNPLCASDGKSYDNACQIKEASCQKQEKIE

Avitag
VMSLGRCQDNTTTTTKSEDGHYARTDYAENANKLE

ESAREHHIPCPEHYNGFCMHGKCEHSINMQEPSCR

CDAGYTGQHCEKKDYSVLYVVPGPVRFQYVGSGSG

SENLYFQGVRSSSDAHKSEVAHRFKDLGEENFKAL

VLIAFAQYLQQSPFEDHVKLVNEVTEFAKTCVADE

SAENCDKSLHTLFGDKLCTVATLRETYGEMADCCA

KQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAF

HDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYK

AAFTECCQAADKAACLLPKLDELRDEGKASSAKQR

LKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVS

KLVTDLTKVHTECCHGDLLECADDRADLAKYICEN

QDSISSKLKECCEKPLLEKSHCIAEVENDEMPADL

PSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARR

HPDYSVVLLLRLAKTYETTLEKCCAAADPECYAKV

FDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALL

VRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEA

KRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCT

ESLVNRRPCFSALEVDETYVPKEFNAETFTFHADI

CTLSEKERQIKKQTALVELVKHKPKATKEQLKAVM

DDFAAFVEKCCKADDKETCFAEEGKKLVAASQAAL

GLGGGSHHHHHHLNDIFEAQKIEWHE

TMEW19
spTMEFF2(230-
NTTTTTKSEDGHYARTDYAENANKLEESAREHHIP

(SEQ ID NO: 580)
320)G3S-H6
CPEHYNGFCMHGKCEHSINMQEPSCRCDAGYTGQH

CEKKDYSVLYVVPGPVRFQYVGGGSHHHHHH

TMEW20
spTMEFF2(230-320)-
NTTTTTKSEDGHYARTDYAENANKLEESAREHHIP

(SEQ ID NO: 581)
G3S-ratIgG1Fc
CPEHYNGFCMHGKCEHSINMQEPSCRCDAGYTGQH

CEKKDYSVLYVVPGPVRFQYVGGGSPRNCGGDCKP

CICTGSEVSSVFIFPPKPKDVLTITLTPKVTCVVV

DISQDDPEVHFSWFVDDVEVHTAQTRPPEEQFNST

FRSVSELPILHQDWLNGRTFRCKVTSAAFPSPIEK

TISKPEGRTQVPHVYTMSPTKEEMTQNEVSITCMV

KGFYPPDIYVEWQMNGQPQENYKNTPPTMDTDGSY

FLYSKLNVKKEKWQQGNTFTCSVLHEGLHNHHTEK

SLSHSPGKGGGSHHHHHH

7-2 Generation of Anti-TMEFF2 Antibodies

Antibody generation using transgenic rats expressing human immunoglobulin loci (OmniRat®) The OmniRat® contains a chimeric human/rat IgH locus (comprising 22 human V_Hs, all human D and J_Hsegments in natural configuration linked to the rat C_Hlocus) together with fully human IgL loci (12 Vκs linked to Jκ-Cκ and 16 Vλs linked to Jκ-Cκ). (see e.g., Osborn, et al. (2013) J Immunol 190(4): 1481-1490). Accordingly, the rats exhibit reduced expression of rat immunoglobulin, and in response to immunization, the introduced human heavy and light chain transgenes undergo class switching and somatic mutation to generate high affinity chimeric human/rat IgG monoclonal antibodies with fully human variable regions. The preparation and use of OmniRat®, and the genomic modifications carried by such rats, is described in WO14/093908.

OmniRats were immunized with the human TMEFF2 construct FS2-EGF-Tev-HSA(C34S)-His-Avitag (TMEW7, SEQ ID NO: 579) and boosted with the construct spTMEFF2 (230-320)G3S-ratIgG1Fc (TMEW20, SEQ ID NO: 581). Following a 89 day immunization regimen, lymph nodes from the rats were harvested and used to generate hybridomas and the hybridoma supernatants were screened for binding to human TMEFF2-FL-ECD-His-Avitag (TMEW1) protein by ELISA and/or SPARCL (Spatial Proximity Analyte Reagent Capture Luminescence). Several supernatants were selected for secondary ELISA and SPARCL screening of binding to TMEFF ECD, FS2-EGF, or EGF domain only TMEFF2. Based on the screening results, several hybridoma clones were sequenced, expressed and characterized for functionality.

Antibody Generation from Phage Display Libraries

TMEFF2 binding Fabs were selected using standard methods from two sets of de novo pIX phage display libraries as described in Shi et al., J Mol Biol 397:385-96, 2010 and WO2009/085462). Briefly, two sets of libraries, referred to as V3.0 and V5.0, were generated by diversifying human scaffolds where germline VH genes IGHV1-69*01, IGHV3-23*01 and IGHV5-51*01 were recombined with the human IGHJ-4 minigene via the H3 loop (IGHJ-6 minigene was also used in V5.0), and human germline VLkappa genes O12 (IGKV1-39*01), L6 (IGKV3-11*01), A27 (IGKV3-20*01), and B3 (IGKV4-1*01) were recombined with the IGKJ-1 minigene to assemble complete VH and VL domains. Positions in the heavy and light chain variable regions around the H1, H2, L1, L2 and L3 loops in frequent contact with protein and peptide antigens were chosen for diversification. Sequence diversity at selected positions was limited to residues occurring at each position in the IGHV or IGLV germline gene families of the respective IGHV or IGLV genes. Diversity at the H3 loop was generated by utilizing short to mid-sized synthetic loops of lengths 7-14 amino acids for V3.0 libraries, and lengths 6-19 amino acids for V5.0 libraries. The amino acid distribution at H3 was designed to mimic the observed variation of amino acids in human antibodies. The scaffolds utilized to generate libraries were named according to their human VH and VL germline gene origin. For both V3.0 and V5.0 sets, each of the three heavy chain libraries were combined with the four germline light chains or germline light chain libraries to generate 12 unique VH:VL combinations for each set of libraries which are used for selection experiments.

V Region Cloning

Total RNA from hybridoma cell lysates of phage were purified using RNeasy 96 kit (Qiagen) following the manufacturer's protocol. The resulting RNA was quantitated using Drop Sense and either stored at −80° C. or used for cDNA synthesis using Invitrogen SuperScript III First-Strand Synthesis System by RT-PCR (Invitrogen). The first strand cDNA synthesis was carried out using gene specific primers annealed to the constant regions of heavy, kappa, and lambda chains, respectively. The RT-PCR reaction mixture comprised of up to 3 μg of purified RNA, gene specific primer, dNTP mix, reaction buffer, 25 mM MgCl₂, DTT, RNaseOUT™ (40 U/μl, Invitrogen), and SuperScript™ III RT (200 U/μl, Invitrogen Cat #18080-051) was incubated at 50° C. for 50 minutes and 85° C. for 5 minutes. The resulting single-stranded cDNA was stored at −20° C., or used directly for PCR amplification. The PCR reaction was carried out using Platinum Pfx polymerase (Invitrogen). The v-region fragments were amplified by forward and reverse primers annealing to the leader sequences and constant regions of heavy, kappa and lambda chains, respectively, using optimized PCR conditions. The resulting PCR fragments were sequenced, the amino acid sequences of the recovered v-regions were codon optimized and cloned into the pUnder-based expression vector carrying the IgG4 constant region with S228P, F234A and L235A mutations (IgG4PAA isotype).

Expi293 Small Scale Transfection and Purification

Select antibodies identified from the immunization campaigns or phage display were cloned and expressed as IgG1PAA and purified via small 2 ml scale. Expi293™ cells (ThermoFisher Scientific) were seeded at 1.25×10⁵-2.25×10⁵viable cells/mL density in Expi293™ Expression Medium and cultured in 125 mL-2 L shake flasks at 37° C., 7% CO2. Cells were sub-cultured when density reached the log phase growth at 3×10⁶-5×10⁶viable cells/mL with a 98-99% viability.

On day of transfection, the viable cell density and percent viability was determined. Cells were transfected at a density of 3×106 viable cells/mL following manufacturer's Transfection protocol (ThermoFisher Publication Number MAN0007814). Culture were harvested on Day 6 post-transfection by centrifugation at 850×G for 15 minutes before purification. Antibodies were purified from the clarified supernatants using mAb Select Sure resin (GE Healthcare) and dialyzed into PBS. Protein concentrations were determined by A280 measurement on the filtrate using a DropSense Instrument (Trinean).

7-3 Characterization of Anti-TMEFF2 Antibodies

Anti-TMEFF2 Antibodies Bind TMEFF2 with High Affinity

Binding of select IgG4PAA anti-TMEFF2 antibodies to TMEFF2 ECD (TMEW1: TMEFF2-FL ECD-His-Avitag) and/or the membrane proximal region (TMEW19: spTMEFF2(230-320)G3S-H6) was assessed using Proteon (TMEB674, TMEB675, TMEB 565 and TMEB570) or Biacore SPR (TMEB762 and TMEB757). The kinetic parameters of binding selected antibodies are show in Table 59. The anti-TMEFF2 antibodies were found to bind both the TMEFF2 ECD and TMEFF2 membrane proximal region with picomolar affinities.

TABLE 59

TMEFF2

Antibody
Antigen
domain
ka (1/Ms)
kd (1/s)
KD (nM)

TMEB674
TMEW1
ECD
2.01E+06
1.62E−04
0.10

TMEB674
TMEW19
MP*
8.37E+05
1.75E−04
0.20

TMEB675
TMEW1
ECD
2.65E+06
1.53E−04
0.10

TMEB675
TMEW19
MP
9.72E+05
1.58E−04
0.20

TMEB565
TMEW1
ECD
3.84E+05
9.13E−06
0.02

TMEB565
TMEW19
MP
7.77E+05
6.39E−06
0.01

TMEB570
TMEW1
ECD
4.64E+05
5.13E−05
0.11

TMEB570
TMEW19
MP
7.62E+05
4.67E−05
0.06

TMEB762
TMEW1
ECD
5.41E+05
1.74E−04
0.32

TMEB757
TMEW1
ECD
5.42E+05
1.67E−04
0.31

*MP: membrane proximal region

ProteOn SPR

The binding of anti-TMEFF2 mAbs to the ECD and the membrane proximal region of human TMEFF2 was measured by ProteOn SPR (Bio-Rad). The purified mAbs (diluted to a final concentration of 1 μg/ml in PBST) were used as ligands in the assay and were immobilized through Fc capture to Goat Anti-Human (GAH) IgG Fc. For amine coupling of the GAH IgG Fc, a 1:1 mixture of EDC (40 mM) and NHS (10 mM) were mixed immediately prior to injection to activate the chip surface and injected in the vertical orientation. GAH-Fc (at 30 μg/ml) antibody in acetate buffer (pH 5.0) was then flowed over the surface for 300 seconds at 30 μl/min in the vertical orientation. Any remaining reactive carboxyl groups in the surface were subsequently deactivated by injecting 1M Ethanolamine (pH 8.5) in the same orientation. The antibodies were used at a concentration of 1 μg/ml for immobilization. The antibodies were flowed over the surface in the horizontal direction. Human TMEFF2 ECD or the membrane proximal region in 3-fold dilution series of 5 concentrations (highest concentration ranging from 100-600 nM) was flowed in as analyte in the vertical orientation to bind to the captured molecules. A buffer sample was also injected in the 6^thchannel in the vertical direction to monitor any drift in the baseline signal. The association and dissociation phases for all concentrations were monitored over 3 minutes and 30 (or 15) minutes respectively, at a flow rate of 100 μL/min. The binding surface was regenerated for the next interaction cycle using an 18 second pulse of 0.8% phosphoric acid to remove the bound antigen. The raw data were processed by subtracting two sets of reference data from the response data: 1) the inter-spot signals to correct for the non-specific interactions between the antigen and the empty chip surface; 2) the empty channel signals (where only PBST was flowed over the chip) to correct for non-specific baseline drift.

Biacore 8K SPR

The binding of anti-TMEFF2 mAbs to human TMEFF2 ECD was measured by Biacore 8K SPR. The format of the assay was to capture the mAbs using a high density anti-human Fc surface, then inject human TMEFF2 concentration titration using a single cycle kinetics method. Goat anti-human Fc IgG (Jackson Immunoresearch, Cat #109-005-098) was directly immobilized via amine coupling at 30 μg/mL in 10 mM acetate buffer, pH 4.5 on flow cells 1 and 2, on CM5 Sensor Chip (GE) with a flow rate of 30 μL/min in HBSP (GE) buffer. The mAbs were captured on the anti-human Fc IgG surface at 0.5 μg/ml (˜200-300 RU) on flow cell 2. The running buffer was then changed to HBSP+100 ug/ml BSA. TMEFF2 ECD at 30 nM concentration in 3-fold dilution series was injected from low to high concentration using single cycle kinetics method. The off-rate was monitored 30 minutes after the last or highest concentration injection and then the surface was regenerated using 0.8% phosphoric acid (Bio-Rad). A buffer blank run, capturing the same mAbs and using the same conditions of sample run was also completed. The raw data were processed by subtracting two sets of reference data from the response data: 1) reference flow cell 1 subtracted from sample flow cell 2 and 2) buffer blank run from experimental run. The processed data at all concentrations for each mAb were globally fit to a 1:1 simple Langmuir binding model to extract estimates of the kinetic (kon, koff) and affinity (KD) constants.

Thermal Stability of Anti-TMEFF2 Antibodies

TMEB675 showed lower than usual thermal stability profile by DSC (Differential Scanning calorimetry) with onset of unfolding Tm=52° C. and the first thermal transition (Tm1) at =60.4° C. A closer examination of the sequence of TMEB675 (see Example 4) showed the presence of Somatic HyperMutations (SHM) within the framework region of heavy and light chain. Several re-engineered variants were sub-cloned, expressed, purified and profiled by DSC. The resulting mAbs TMEB762 and TMEFB757 showed desirable thermal stability profile (Tm1=69.4° C. and Tm1=69.7° C. respectively). In comparison to TMEB675, TMEB762 had the following amino acid modifications in the heavy chain: R14P, P20L and H81Q, while TMEFB757 had the following amino acid modifications in the heavy chain: R14P and P20L. In comparison to TMEB675, TMEB762 had the following amino acid modifications in the light chain: MD and A91P, while TMEFB757 had A91P modification in the light chain. Residue numbering is according to Kabat. The kinetic parameters of binding of TMEB675, TMEB762 to TMEFF2 ECD is shown in Table 59.

7-4 Structural Characterization of Anti-TMEFF2 Antibodies

The cDNA sequences and amino acid translations of the antibodies were obtained using standard techniques. After polypeptide sequence determination, some antibody cDNAs encoding the variable regions or full-length antibodies were codon optimized using standard methods for scale-up expression.

Table 60 shows the HCDR1 and the HCDR2 amino acid sequences of select anti-TMEFF2 antibodies.

Table 61 shows the HCDR3 amino acid sequences of select anti-TMEFF2 antibodies.

Table 62 shows the LCDR1 and the LCDR2 amino acid sequences of select anti-TMEFF2 antibodies.

Table 63 shows the LCDR3 amino acid sequences of select anti-TMEFF2 antibodies.

Table 64 shows the VH and the VL amino acid sequence of select anti-TMEFF2 antibodies.

Table 65 shows the SEQ ID NOs: of heavy and light chains of select anti-TMEFF2 antibodies.

Table 66 shows the heavy chain amino acid sequences of select anti-TMEFF2 antibodies.

Table 67 shows the light chain amino acid sequences of select anti-TMEFF2 antibodies.

Table 68 shows the SEQ ID NOs: of polynucleotides encoding various anti-TMEFF2 antibody chains.

TABLE 60

HCDR1

HCDR2

HCDR1
SEQ ID

SEQ ID

mAb
sequence
NO:
HCDR2 sequence
NO:

TMEB675
SYSMS
582
VISGSGGFTDYADSVKG
584

TMEB570
SYYIS
583
GIIPISGRANYAQKFQG
585

TMEB674
SYSMS
582
VISGGGSFTSYADSVKG
586

TMEB565
SYYIS
583
GIIPISGRANYAQKFQG
585

TMEB762
SYSMS
582
VISGSGGFTDYADSVKG
584

TMEB757
SYSMS
582
VISGSGGFTDYADSVKG
584

TABLE 61

mAb
HCDR3 sequence
HCDR3 SEQ ID

TMEB675
MPLNSPHDY
587

TMEB570
DGYSSGRSTTYAFDY
16

TMEB674
MPLNSPHDC
17

TMEB565
DGYSSGRSTTYAFDY
16

TMEB762
MPLNSPHDY
587

TMEB757
MPLNSPHDY
587

TABLE 62

LCDR1

LCDR2

LCDR1 amino
SEQ ID
LCDR2
SEQ ID

mAb
acid
NO:
amino acid
NO:

TMEB675
RASQGIRNDLG
18
AASSLQS
588

TMEB570
RASQSVSTYYLA
19
GASYRAT
21

TMEB674
RASQGIRNDLG
18
AASSLQS
588

TMEB565
RASQGIRNDLG
18
AASSLQS
588

TMEB762
RASQGIRNDLG
18
AASSLQS
588

TMEB757
RASQGIRNDLG
18
AASSLQS
588

TABLE 63

mAb
LCDR3 amino acid
LCDR3 SEQ ID NO:

TMEB675
LQDYNYALT
22

TMEB570
QQYGHSPIT
23

TMEB674
LQDYNYSLT
24

TMEB565
LQDYNYALT
22

TMEB762
LQDYNYPLT
603

TMEB757
LQDYNYPLT
603

TABLE 64

VH amino
VH SEQ

VL amino
VL SEQ

Antibody
VH name
acid sequence
ID NO:
VL name
acid sequence
ID NO:

TMEB675
TMEH411
EVQLLESGGGLVQRGGSLRP
25
TMEL127
AIQMTQSPSSLSASVGD
28

SCAASGFTFSSYSMSWVRQA

RVTITCRASQGIRNDLG

PGKGLEWVSVISGSGGFTDY

WYQQKPGKAPKLLIYAA

ADSVKGRFTISRDNSKNTLY

SSLQSGVPSRFSGSGSG

LHMNSLRAEDTAVYYCARMP

TDFTLTISSLQPEDFAT

LNSPHDYWGQGTLVTVSS

YYCLQDYNYALTFGGGT

KVEIK

TMEB570
TMEH396
QVQLVQSGAEVKKPGSSVKV
589
TMEL112
EIVLTQSPGTLSLSPGE
29

SCKASGGTFSSYYISWVRQA

RATLSCRASQSVSTYYL

PGQGLEWMGGIIPISGRANY

AWYQQKPGQAPRLLIYG

AQKFQGRVTITADESTSTAY

ASYRATGIPDRFSGSGS

MELSSLRSEDTAVYYCARDG

GTDFTLTISRLEPEDFA

YSSGRSTTYAFDYWGQGTLV

VYYCQQYGHSPITFGQG

TVSS

TKVEIK

TMEB674
TMEH410
EVQLLESGGLVQPPGGSLRL
27
TMEL126
AIQMTQSPSSLSASVGD
30

SCAASGFTFSSYSMSWVRQA

RVTITCRASQGIRNDLG

PGKGLEWVSVISGGGSFTSY

WYQQKPGKAPKLLIYAA

ADSVKGRFTISRDNSNNTLY

SSLQSGVPSRFSGSGSG

LQMSSLRAEDTAFYYCARMP

TDFTLTISSLQPEDFAT

LNSPHDCWGQGTLVTVSS

YYCLQDYNYSLTFGGGT

KVEIR

TMEB565
TMEH396
QVQLVQSGAEVKKPGSSVKV
589
TMEL111
EIVLTQSPGTLSLSPGE
31

SCKASGGTFSSYYISWVRQA

RATLSCRASQSVATYYL

PGQGLEWMGGIIPISGRANY

AWYQQKPGQAPRLLIYG

AQKFQGRVTITADESTSTAY

ASSRATGIPDRFSGSGS

MELSSLRSEDTAVYYCARDG

GTDFTLTISRLEPEDFA

YSSGRSTTYAFDYWGQGTLV

VYYCQQYGYNPITFGQG

TVSS

TKVEIK

TMEB762
TMEH459
EVQLLESGGGLVQPGGSLRL
604
DL3L129
DIQMTQSPSSLSASVGD
607

SCAASGFTFSSYSMSWVRQA

RVTITCRASQGIRNDLG

PGKGLEWVSVISGSGGFTDY

WYQQKPGKAPKLLIYAA

ADSVKGRFTISRDNSKNTLY

SSLQSGVPSRFSGSGSG

LQMNSLRAEDTAVYYCARMP

TDFTLTISSLQPEDFAT

LNSPHDYWGQGTLVTVSS

YYCLQDYNYPLTFGGGT

KEVIK

TMEB757
TMEH460
EVQLLESGGGLVQPGGSLRL
612
B76L85
AIQMTQSPSSLSASVGD
613

SCAASGFTFSSYSMSWVRQA

RVTITCRASQGIRNDLG

PGKGLEWVSVISGSGGFTDY

WYQQKPGKAPKLLIYAA

ADSVKGRFTISRDNSKNTLY

SSLQSGVPSRFSGSGSG

LHMNSLRAEDTAVYYCARMP

TDFTLTISSLQPEDFAT

LNSPHDYWGQGTLVTVSS

YYCLQDYNYPLTFGGGT

KVEIK

TABLE 65

HC protein SEQ
LC protein SEQ

Antibody
VH name
VL name
ID NO:
ID NO:

TMEB675
TMEH411
TMEL127
32
35

TMEB570
TMEH396
TMEL112
33
36

TMEB674
TMEH410
TMEL126
34
37

TMEB565
TMEH396
TMEL111
33
38

TMEB762
TMEH459
DL3L129
614
615

TMEB757
TMEH460
B76L85
616
617

TABLE 66

HC protein

SEQ ID NO:
HC amino acid sequence

32
evqllesggglvqrggslrpscaasgftfssysmsw

(TMEB675
vrqapgkglewvsvisgsggftdyadsvkgrftisr

HC)
dnskntlylhmnslraedtavyycarmplnsphdyw

gqgtlvtvssastkgpsvfplapcsrstsestaalg

clvkdyfpepvtvswnsgaltsgvhtfpavlqssgl

yslssvvtvpssslgtktytcnvdhkpsntkvdkrv

eskygppcppcpapeaaggpsvflfppkpkdtlmis

rtpevtcvvvdvsqedpevqfnwyvdgvevhnaktk

preeqfnstyrvvsyltvlhqdwlngkeykckvsnk

glpssiektiskakgqprepqvytlppsqeemtknq

vsltclvkgfypsdiavewesngqpennykttppvl

dsdgsfflysrltvdksrwqegnvfscsvmhealhn

hytqkslslslgk

33
qvqlvqsgaevkkpgssvkvsckasggtfssyyisw

(TMEB570,
vrqapgqglewmggiipisgranyaqkfqgrvtita

TMEB565
deststaymelsslrsedtavyycardgyssgrstt

HC)
yafdywgqgtlvtvssastkgpsvfplapcsrstse

staalgclvkdyfpepvtvswnsgaltsgvhtfpav

lqssglyslssvvtvpssslgtktytcnvdhkpsnt

kvdkrveskygppcppcpapeaaggpsvflfppkpk

dtlmisrtpevtcvvvdvsqedpevqfnwyvdgvev

hnaktkpreeqfnstyrvvsyltvlhqdwlngkeyk

ckvsnkglpssiektiskakgqprepqvytlppsqe

emtknqvsltclvkgfypsdiavewesngqpennyk

ttppvldsdgsfflysrltvdksrwqegnvfscsvm

healhnhytqkslslslgk

34
evqllesggglvqppggslrlscaasgftfssysms

(TMEB674
wvrqapgkglewvsvisgggsftsyadsvkgrftis

HC)
rdnsnntlylqmsslraedtafyycarmplnsphdc

wgqgtlvtvssastkgpsvfplapcsrstsestaal

gclvkdyfpepvtvswnsgaltsgvhtfpavlqssg

lyslssvvtvpssslgtktytcnvdhkpsntkvdkr

veskygppcppcpapeaaggpsvflfppkpkdtlmi

srtpevtcvvvdvsqedpevqfnwyvdgvevhnakt

kpreeqfnstyrvvsyltvlhqdwlngkeykckvsn

kglpssiektiskakgqprepqvytlppsqeemtkn

qvsltclvkgfypsdiavewesngqpennykttppv

ldsdgsfflysrltvdksrwqegnvfscsvmhealh

nhytqkslslslgk

614
evqllesggglvqpggslrlscaasgftfssysmsw

(TMEB762
vrqapgkglewvsvisgsggftdyadsvkgrftisr

HC)
dnskntlylqmnslraedtavyycarmplnsphdyw

gqgtlvtvssastkgpsvfplapcsrstsestaalg

clvkdyfpepvtvswnsgaltsgvhtfpavlqssgl

yslssvvtvpssslgtktytcnvdhkpsntkvdkrv

eskygppcppcpapeaaggpsvflfppkpkdtlmis

rtpevtcvvvdvsqedpevqfnwyvdgvevhnaktk

preeqfnstyrvvsyltvlhqdwlngkeykckvsnk

glpssiektiskakgqprepqvytlppsqeemtknq

vsltclvkgfypsdiavewesngqpennykttppvl

dsdgsfflysrltvdksrwqegnvfscsvmhealhn

hytqkslslslgk

616
evqllesggglvqpggslrlscaasgftfssysmsw

(TMEB757
vrqapgkglewvsvisgsggftdyadsvkgrftisr

HC)
dnskntlylhmnslraedtavyycarmplnsphdyw

gqgtlvtvssastkgpsvfplapcsrstsestaalg

clvkdyfpepvtvswnsgaltsgvhtfpavlqssgl

yslssvvtvpssslgtktytcnvdhkpsntkvdkrv

eskygppcppcpapeaaggpsvflfppkpkdtlmis

rtpevtcvvvdvsqedpevqfnwyvdgvevhnaktk

preeqfnstyrvvsyltvlhqdwlngkeykckvsnk

glpssiektiskakgqprepqvytlppsqeemtknq

vsltclvkgfypsdiavewesngqpennykttppvl

dsdgsfflysrltvdksrwqegnvfscsvmhealhn

hytqkslslslgk

TABLE 67

LC protien

SEQ ID NO:
LC amino acid sequence

35
aiqmtqspsslsasvgdrvtitcrasqgirndlgwyq

(TMEB675
qkpgkapklliyaasslqsgvpsrfsgsgsgtdftlt

LC)
isslqpedfatyyclqdynyaltfgggtkveikrtva

apsvfifppsdeqlksgtasvvcllnnfypreakvqw

kvdnalqsgnsqesvteqdskdstyslsstltlskad

yekhkvyacevthqglsspvtksfnrgec

36
eivltqspgtlslspgeratlscrasqsvstyylawy

(TMEB570
qqkpgqpaprlliygasyratgipdrfsgsgsgtdft

LC)
ltisrlepedfavyycqqyghspitfgqgtkveikrt

vaapsvfifppsdeqlksgtasvvcllnnfypreakv

qwkvdnalqsgnsqesvteqdskdstyslsstltlsk

adyekhkvyacevthqglsspvtksfnrgec

37
aiqmtqspsslsasvgdrvtitcrasqgirndlgwyq

(TMEB674
qkpgkapklliyaallsqsgvpsrfsgsgsgtdftlt

LC)
isslqpedfatyyclqdynysltfgggtkveirrtva

apsvfifppsdeqlksgtasvvcllnnfypreakvqw

kvdnalqsgnsqesvteqdskdstyslsstltlskad

yekhkvyacevthqglsspvtksfnrgec

38
eivltqspgtlslspgeratlscrasqsvatyylawy

(TMEB565
qqkpgqaprlliygassratgipdrfsgsgsgtdftl

LC)
tisrlepedfavyycqqygynpitfgqgtkveikrtv

aapsvfifppsdeqlksgtasvvcllnnfypreakvq

wkvdnalqsgnsqesvteqdskdstyslsstltlska

dyekhkvyacevthqglsspvtksfnrgec

615
diqmtqspsslsasvgdrvtitcrasqgirndlgwyq

(TMEB762
qkpgkapklliyaasslqsgvpsrfsgsgsgtdftlt

LC)
isslqpedfatyyclqdynypltfgggtkveikrtva

apsvfifppsdeqlksgtasvvcllnnfypreakvqw

vdnalqsgnsqesvteqdskdstyslsstltlskady

ekhkvyacevthqglsspvtksfnrgec

617
aikmtqspsslsasvgdrvtitcrasqgirndlgwyq

(TMEB757
qkpgkapklliyaasslqsgvpsrfsgsgsgtdftlt

LC)
isslqpedfatyyclqdynypltfgggtkveikrtva

apsvfifppsdeqlksgtasvvcllnnfypreakvqw

kvdnalqsgnsqesvteqdskdstyslsstltlskad

yekhkvyacevthqglsspvtksfnrgec

TABLE 68

VH
VL
HC
LC

cDNA
cDNA
cDNA
cDNA

SEQ ID
SEQ ID
SEQ ID
SEQ ID

Antibody
NO:
NO:
NO:
NO:

TMEB675
39
42
46
49

TMEB570
40
43
47
50

TMEB674
41
44
48
51

TMEB565
40
45
47
590

TMEB762
618
619
620
621

TMEB757
622
623
624
625

(TMEB675 VH cDNA)

SEQ ID NO: 39

Gaggtgcagctgctggaaageggcggaggcctggtgcagagaggaggaagcctgagacccagctgtgccgccagcggcttcaccttc

agcagctacagcatgagctgggtcaggcaggcccctggcaaaggactggagtgggtgagcgtgattagcggcagcggcggcttcacc

gattacgccgacagcgtgaagggcaggttcaccatcagcagggacaatagcaagaacaccctgtacctgcacatgaacagcctgagg

gccgaggacaccgccgtgtactactgcgccaggatgcccctgaacagccctcacgactactggggccagggaaccctggtgaccgtg

tccagc

(TMEB570, TMEB565 VH cDNA)

SEQ ID NO: 40

Caggtgcagctggtgcagagcggcgcggaagtgaaaaaaccgggcagcagcgtgaaagtgagctgcaaagcgagcggcggcaccttc

agctcctattacattagctgggtgcgccaggcgccgggccagggcctggaatggatgggtggcattatcccaatcagtgggcgtgct

aattatgcgcagaaatttcagggccgcgtgaccattaccgctgatgaaagcaccagcaccgcgtatatggaactgagcagcctgcgc

agcgaagataccgcggtgtattattgcgcgcgcgacggctacagtagtggacgtagcacaacatacgcatttgactattggggccag

ggcaccctggtgaccgtgtcgagt

(TMEB674 VH cDNA)

SEQ ID NO: 41

Gaagtgcagctgctggagagcggaggaggactggtgcagcctcctggcggaagcctgagactgagctgcgccgctagcggcttcacc

ttcagcagctacagcatgagctgggtgagacaggctcctggcaagggcctggagtgggtgagcgtgatcagcggcggaggcagcttt

accagctacgccgacagcgtgaagggcaggttcaccatcagcagggacaacagcaacaacaccctgtacctgcagatgagcagcctg

agggccgaggacaccgccttctactactgcgccaggatgcccctgaacagcccccatgactgctggggacagggcaccctggtgacc

gtgagcagc

(TMEB675 VL cDNA)

SEQ ID NO: 42

Gccatccagatgacccagagccctagcagcctgagcgctagcgtgggcgacagggtgaccatcacctgcagggccagccagggcatc

agaaacgacctgggctggtaccagcagaagcccggcaaggcccccaagctgctgatctacgccgccagcagcctgcagagcggagtg

cctagcaggttcagcggaagcggcagcggcaccgacttcaccctgaccatcagcagcctgcagcccgaggacttcgccacctactac

tgcctgcaggactacaactacgccctgacattcggcggcggcaccaaggtggagatcaag

(TMEB570 VL cDNA)

SEQ ID NO: 43

Gaaattgtgctgacccagagcccgggcaccctgagcctgagcccgggcgaacgcgcgaccctgagctgccgcgcgagccagagcgtt

tccacatactacctggcgtggtatcagcagaaaccgggccaggcgccgcgcctgctgatttacggtgcctcctatcgcgcgaccggc

attccggatcgctttagcggcagcggttccggcaccgattttaccctgaccattagccgcctggaaccggaagattttgcggtgtat

tattgccagcagtacggtcacagcccgattacttttggccagggcaccaaagtggaaatcaaa

(TMEB674 VL cDNA)

SEQ ID NO: 44

Gccatccagatgacccagagccctagcagcctgagcgctagcgtgggcgacagggtgaccatcacctgcagggccagccagggcatc

aggaacgacctgggctggtaccagcagaagcccggcaaggcccccaagctgctgatctacgccgccagcagcctgcagagcggagtg

cctagcaggttcagcggcagcggaagcggcaccgacttcaccctgaccatctccagcctgcagcccgaggacttcgccacctactac

tgcctgcaggactacaactacagcctgaccttcggcggcggcaccaaggtggagatcagg

(TMEB565 VL cDNA)

SEQ ID NO: 45

Gaaattgtgctgacccagagcccgggcaccctgagcctgagcccgggcgaacgcgcgaccctgagctgccgcgcgagccagagcgtt

gccacctattatcttgcgtggtatcagcagaaaccgggccaggcgccgcgcctgctgatttacggtgcatcctcccgtgcgaccggc

attccggatcgctttagcggcagcggttccggcaccgattttaccctgaccattagccgcctggaaccggaagattttgcggtgtat

tattgccagcagtacggctataacccaattacctttggccagggcaccaaagtggaaatcaaa

(TMEB675 HC cDNA)

SEQ ID NO: 46

gaggtgcagctgctggaaagcggcggaggcctggtgcagagaggaggaagcctgagacccagctgtgccgccagcggcttcaccttc

agcagctacagcatgagctgggtcaggcaggcccctggcaaaggactggagtgggtgagcgtgattagcggcagcggcggcttcacc

gattacgccgacagcgtgaagggcaggttcaccatcagcagggacaatagcaagaacaccctgtacctgcacatgaacagcctgagg

gccgaggacaccgccgtgtactactgcgccaggatgcccctgaacagccctcacgactactggggccagggaaccctggtgaccgtg

tccagcgcttccaccaagggcccatccgtcttccccctggcgccctgctccaggagcacctccgagagcacagccgccctgggctgc

ctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtc

ctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacgaaaacctacacctgcaacgta

gatcacaagcccagcaacaccaaggtggacaagagagttgagtccaaatatggtcccccatgcccaccatgcccagcacctgaggcc

gccgggggaccatcagtcttcctgttccccccaaaacccaaggacactctcatgatctcccggacccctgaggtcacgtgcgtggtg

gtggacgtgagccaggaagaccccgaggtccagttcaactggtacgtggatggcgtggaggtgcataatgccaagacaaagccgcgg

gaggagcagttcaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaacggcaaggagtacaagtgc

aaggtctccaacaaaggcctcccgtcctccatcgagaaaaccatctccaaagccaaagggcagccccgagagccacaggtgtacacc

ctgcccccatcccaggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctaccccagcgacatcgccgtg

gagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagc

aggctaaccgtggacaagagcaggtggcaggaggggaatgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacaca

cagaagagcctctccctgtctctgggtaaa

(TMEB570, TMEB565 HC cDNA)

SEQ ID NO: 47

caggtgcagctggtgcagagcggcgcggaagtgaaaaaaccgggcagcagcgtgaaagtgagctgcaaagcgagcggcggcaccttc

agctcctattacattagctgggtgcgccaggcgccgggccagggcctggaatggatgggtggcattatcccaatcagtgggcgtgct

aattatgcgcagaaatttcagggccgcgtgaccattaccgctgatgaaagcaccagcaccgcgtatatggaactgagcagcctgcgc

agcgaagataccgcggtgtattattgcgcgcgcgacggctacagtagtggacgtagcacaacatacgcatttgactattggggccag

ggcaccctggtgaccgtgtcgagtgcttccaccaagggcccatccgtcttccccctggcgccctgctccaggagcacctccgagagc

acagccgccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtg

cacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacgaaa

acctacacctgcaacgtagatcacaagcccagcaacaccaaggtggacaagagagttgagtccaaatatggtcccccatgcccacca

tgcccagcacctgaggccgccgggggaccatcagtcttcctgttccccccaaaacccaaggacactctcatgatctcccggacccct

gaggtcacgtgcgtggtggtggacgtgagccaggaagaccccgaggtccagttcaactggtacgtggatggcgtggaggtgcataat

gccaagacaaagccgcgggaggagcagttcaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaac

ggcaaggagtacaagtgcaaggtctccaacaaaggcctcccgtcctccatcgagaaaaccatctccaaagccaaagggcagccccga

gagccacaggtgtacaccctgcccccatcccaggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctac

cccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggc

tccttcttcctctacagcaggctaaccgtggacaagagcaggtggcaggaggggaatgtcttctcatgctccgtgatgcatgaggct

ctgcacaaccactacacacagaagagcctctccctgtctctgggtaaa

(TMEB674 HC cDNA)

SEQ ID NO: 48

gaagtgcagctgctggagagcggaggaggactggtgcagcctcctggcggaagcctgagactgagctgcgccgctagcggatcacct

tcagcagctacagcatgagctgggtgagacaggctcctggcaagggcctggagtgggtgagcgtgatcagcggcggaggcagattac

cagctacgccgacagcgtgaagggcaggttcaccatcagcagggacaacagcaacaacaccctgtacctgcagatgagcagcctgag

ggccgaggacaccgccttctactactgcgccaggatgcccctgaacagcccccatgactgctggggacagggcaccctggtgaccgt

gagcagcgcttccaccaagggcccatccgtcttccccctggcgccctgctccaggagcacctccgagagcacagccgccctgggctg

cctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgt

cctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacgaaaacctacacctgcaacgt

agatcacaagcccagcaacaccaaggtggacaagagagttgagtccaaatatggtcccccatgcccaccatgcccagcacctgaggc

cgccgggggaccatcagtcttcctgttccccccaaaacccaaggacactctcatgatctcccggacccctgaggtcacgtgcgtggt

ggtggacgtgagccaggaagaccccgaggtccagttcaactggtacgtggatggcgtggaggtgcataatgccaagacaaagccgcg

ggaggagcagttcaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaacggcaaggagtacaagtg

caaggtctccaacaaaggcctcccgtcctccatcgagaaaaccatctccaaagccaaagggcagccccgagagccacaggtgtacac

cctgcccccatcccaggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctaccccagcgacatcgccgt

ggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacag

caggctaaccgtggacaagagcaggtggcaggaggggaatgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacac

acagaagagcctctccctgtctctgggtaaa

(TMEB675 LC cDNA)

SEQ ID NO: 49

Gccatccagatgacccagagccctagcagcctgagcgctagcgtgggcgacagggtgaccatcacctgcagggccagccagggcatc

agaaacgacctgggctggtaccagcagaagcccggcaaggcccccaagctgctgatctacgccgccagcagcctgcagagcggagtg

cctagcaggttcagcggaagcggcagcggcaccgacttcaccctgaccatcagcagcctgcagcccgaggacttcgccacctactac

tgcctgcaggactacaactacgccctgacattcggcggcggcaccaaggtggagatcaagcgtacggtggctgcaccatctgtcttc

atcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaa

gtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagc

ctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcg

cccgtcacaaagagcttcaacaggggagagtgt

(TMEB570 LC cDNA)

SEQ ID NO: 50

Gaaattgtgctgacccagagcccgggcaccctgagcctgagcccgggcgaacgcgcgaccctgagctgccgcgcgagccagagcgtt

tccacatactacctggcgtggtatcagcagaaaccgggccaggcgccgcgcctgctgatttacggtgcctcctatcgcgcgaccggc

attccggatcgctttagcggcagcggttccggcaccgattttaccctgaccattagccgcctggaaccggaagattttgcggtgtat

tattgccagcagtacggtcacagcccgattacttttggccagggcaccaaagtggaaatcaaacgtacggtggctgcaccatctgtc

ttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggcc

aaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctac

agcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagc

tcgcccgtcacaaagagcttcaacaggggagagtgt

(TMEB674 LC cDNA)

SEQ ID NO: 51

Gccatccagatgacccagagccctagcagcctgagcgctagcgtgggcgacagggtgaccatcacctgcagggccagccagggcatc

aggaacgacctgggctggtaccagcagaagcccggcaaggcccccaagctgctgatctacgccgccagcagcctgcagagcggagtg

cctagcaggttcagcggcagcggaagcggcaccgacttcaccctgaccatctccagcctgcagcccgaggacttcgccacctactac

tgcctgcaggactacaactacagcctgaccttcggcggcggcaccaaggtggagatcaggcgtacggtggctgcaccatctgtcttc

atcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaa

gtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagc

ctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcg

cccgtcacaaagagcttcaacaggggagagtgt

(TMEB565 LC cDNA)

SEQ ID NO: 590

Gaaattgtgctgacccagagcccgggcaccctgagcctgagcccgggcgaacgcgcgaccctgagctgccgcgcgagccagagcgtt

gccacctattatcttgcgtggtatcagcagaaaccgggccaggcgccgcgcctgctgatttacggtgcatcctcccgtgcgaccggc

attccggatcgctttagcggcagcggttccggcaccgattttaccctgaccattagccgcctggaaccggaagattttgcggtgtat

tattgccagcagtacggctataacccaattacctttggccagggcaccaaagtggaaatcaaacgtacggtggctgcaccatctgtc

ttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggcc

aaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctac

agcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagc

tcgcccgtcacaaagagcttcaacaggggagagtgt

(TMEB762 VH cDNA)

SEQ ID NO: 618

gaggtgcagctgctggaaagcggcggaggcctggtgcagcccggaggaagcctgagactcagctgtgccgccagcggcttcaccttc

agcagctacagcatgagctgggtcaggcaggcccctggcaaaggactggagtgggtgagcgtgattagcggcagcggcggcttcacc

gattacgccgacagcgtgaagggcaggttcaccatcagcagggacaatagcaagaacaccctgtacctgcagatgaacagcctgagg

gccgaggacaccgccgtgtactactgcgccaggatgcccctgaacagccctcacgactactggggccagggaaccctggtgaccgtg

tccagc

(TMEB762 VL cDNA)

SEQ ID NO: 619

gacatccagatgacccagagccctagcagcctgagcgctagcgtgggcgacagggtgaccatcacctgcagggccagccagggcatc

agaaacgacctgggctggtaccagcagaagcccggcaaggcccccaagctgctgatctacgccgccagcagcctgcagagcggagtg

cctagcaggttcagcggaagcggcagcggcaccgacttcaccctgaccatcagcagcctgcagcccgaggacttcgccacctactac

tgcctgcaggactacaactaccccctgacattcggcggcggcaccaaggtggagatcaag

(TMEB762 HC cDNA)

SEQ ID NO: 620

gaggtgcagctgctggaaagcggcggaggcctggtgcagcccggaggaagcctgagactcagctgtgccgccagcggcttcaccttc

agcagctacagcatgagctgggtcaggcaggcccctggcaaaggactggagtgggtgagcgtgattagcggcagcggcggcttcacc

gattacgccgacagcgtgaagggcaggttcaccatcagcagggacaatagcaagaacaccctgtacctgcagatgaacagcctgagg

gccgaggacaccgccgtgtactactgcgccaggatgcccctgaacagccctcacgactactggggccagggaaccctggtgaccgtg

tccagcgcttccaccaagggcccatccgtcttccccctggcgccctgctccaggagcacctccgagagcacagccgccctgggctgc

ctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtc

ctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacgaaaacctacacttgcaacgta

gatcacaagcccagcaacaccaaggtggacaagagagttgagtccaaatatggtcccccatgcccaccatgcccagcacctgaggcc

gccgggggaccatcagtcttcctgttccccccaaaacccaaggacactctcatgatctcccggacccctgaggtcacgtgcgtggtg

gtggacgtgagccaggaagaccccgaggtccagttcaactggtacgtggatggcgtggaggtgcataatgccaagacaaagccgcgg

gaggagcagttcaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaacggcaaggagtacaagtgc

aaggtctccaacaaaggcctcccgtcctccatcgagaaaaccatctccaaagccaaagggcagccccgagagccacaggtgtacacc

ctgcccccatcccaggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctaccccagcgacatcgccgtg

gagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagc

aggctaaccgtggacaagagcagatggcaggaggggaatgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacaca

cagaagagcctctccctgtctctgggtaaa

(TMEB762 LC cDNA)

SEQ ID NO: 621

gacatccagatgacccagagccctagcagcctgagcgctagcgtgggcgacagggtgaccatcacctgcagggccagccagggcatc

agaaacgacctgggctggtaccagcagaagcccggcaaggcccccaagctgctgatctacgccgccagcagcctgcagagcggagtg

cctagcaggttcagcggaagcggcagcggcaccgacttcaccctgaccatcagcagcctgcagcccgaggacttcgccacctactac

tgcctgcaggactacaactaccccctgacattcggcggcggcaccaaggtggagatcaagcgtacggtggctgcaccatctgtcttc

atcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaa

gtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagc

ctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcg

cccgtcacaaagagcttcaacaggggagagtgttag

(TMEB757 VH cDNA)

SEQ ID NO: 622

gaggtgcagctgctggaaagcggcggaggcctggtgcagcccggaggaagcctgagactcagctgtgccgccagcggcttcaccttc

agcagctacagcatgagctgggtcaggcaggcccctggcaaaggactggagtgggtgagcgtgattagcggcagcggcggcttcacc

gattacgccgacagcgtgaagggcaggttcaccatcagcagggacaatagcaagaacaccctgtacctgcacatgaacagcctgagg

gccgaggacaccgccgtgtactactgcgccaggatgcccctgaacagccctcacgactactggggccagggaaccctggtgaccgtg

tccagc

(TMEB757 VL cDNA)

SEQ ID NO: 623

gccatccagatgacccagagccctagcagcctgagcgctagcgtgggcgacagggtgaccatcacctgcagggccagccagggcatc

agaaacgacctgggctggtaccagcagaagcccggcaaggcccccaagctgctgatctacgccgccagcagcctgcagagcggagtg

cctagcaggttcagcggaagcggcagcggcaccgacttcaccctgaccatcagcagcctgcagcccgaggacttcgccacctactac

tgcctgcaggactacaactaccccctgacattcggcggcggcaccaaggtggagatcaag

(TMEB757 HC cDNA)

SEQ ID NO: 624

gaggtgcagctgctggaaagcggcggaggcctggtgcagcccggaggaagcctgagactcagctgtgccgccagcggcttcaccttc

agcagctacagcatgagctgggtcaggcaggcccctggcaaaggactggagtgggtgagcgtgattagcggcagcggcggcttcacc

gattacgccgacagcgtgaagggcaggttcaccatcagcagggacaatagcaagaacaccctgtacctgcacatgaacagcctgagg

gccgaggacaccgccgtgtactactgcgccaggatgcccctgaacagccctcacgactactggggccagggaaccctggtgaccgtg

tccagcgcttccaccaagggcccatccgtcttccccctggcgccctgctccaggagcacctccgagagcacagccgccctgggctgc

ctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtc

ctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacgaaaacctacacttgcaacgta

gatcacaagcccagcaacaccaaggtggacaagagagttgagtccaaatatggtcccccatgcccaccatgcccagcacctgaggcc

gccgggggaccatcagtcttcctgttccccccaaaacccaaggacactctcatgatctcccggacccctgaggtcacgtgcgtggtg

gtggacgtgagccaggaagaccccgaggtccagttcaactggtacgtggatggcgtggaggtgcataatgccaagacaaagccgcgg

gaggagcagttcaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaacggcaaggagtacaagtgc

aaggtctccaacaaaggcctcccgtcctccatcgagaaaaccatctccaaagccaaagggcagccccgagagccacaggtgtacacc

ctgcccccatcccaggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctaccccagcgacatcgccgtg

gagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagc

aggctaaccgtggacaagagcagatggcaggaggggaatgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacaca

cagaagagcctctccctgtctctgggtaaa

(TMEB757 LC cDNA)

SEQ ID NO: 625

gccatccagatgacccagagccctagcagcctgagcgctagcgtgggcgacagggtgaccatcacctgcagggccagccagggcatc

agaaacgacctgggctggtaccagcagaagcccggcaaggcccccaagctgctgatctacgccgccagcagcctgcagagcggagtg

cctagcaggttcagcggaagcggcagcggcaccgacttcaccctgaccatcagcagcctgcagcccgaggacttcgccacctactac

tgcctgcaggactacaactaccccctgacattcggcggcggcaccaaggtggagatcaagcgtacggtggctgcaccatctgtcttc

atcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaa

gtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagc

ctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcg

cccgtcacaaagagcttcaacaggggagagtgttag

The frameworks for the select anti-TMEFF2 antibodies are shown in Table 69.

TABLE 69

VH

VL

VH
framework
VL
framework

Antibody
framework
SEQ ID NO:
framework
SEQ ID NO:

TMEB675
VH3_3-23
53
VKI_L11
55

TMEB570
VH1_1-69
54
VKIII_A27
591

TMEB674
VH3_3-23
53
VKI_L11
55

TMEB565
VH1_1-69
54
VKI_L11
55

TMEB762
VH3_3-23
53
VKI_L11
55

TMEB757
VH3_3-23
53
VKI_L11
55

(VH3_3-23 framework)

SEQ ID NO: 53

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVS

AISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK

WGQGTLVTVSS

(VH1_1-69 framework)

SEQ ID NO: 54

QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMG

GIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAR

(VKI_L11 framework)

SEQ ID NO: 55

aiqmtqspsslsasvgdrytitcrasqgirndlgwyqqkpgkapklliy

aasslqsgypsrfsgsgsgtdftltisslqpedfatyyclqdynyp

(VKIII_A27 framework)

SEQ ID NO: 591

EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLI

YGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSP

7-5 Epitope Mapping of Anti-TMEFF2 Antibodies

Epitope mapping of TMEB570 and TMEB675 was done using H/D exchange. TMEW1 (SEQ ID NO: 578) was used as source of TMEFF2 in these assays.

10 μg of TMEW1 in 130 μL of control buffer (50 mM phosphate, 100 mM sodium chloride at pH 7.4) was denatured by adding 130 μL of 4 M guanidine hydrochloride, 0.85 M TCEP buffer (final pH was 2.5) and incubating the mixture for 3 min at 10° C. The mixture was then subjected to on-column pepsin/protease XIII digestion using an in-house packed pepsin/protease XIII (w/w, 1:1) column (2.1×30 mm). The resultant peptides were analyzed using an UPLC-MS system comprised of a Waters Acquity UPLC coupled to a Q Exactive™ Hybrid Quadrupole-Orbitrap Mass Spectrometer (Thermo). The peptides were separated on a 50×1 mm C8 column with a 16.5 min gradient from 2-34% solvent B (0.2% formic acid in acetonitrile). Solvent A was 0.2% formic acid in water. The injection valve and pepsin/protease XIII column and their related connecting tubings were kept inside a cooling box maintained at 10° C. The second switching valve, C8 column and their related connecting stainless steel tubings were inside another chilled circulating box maintained at −6° C. Peptide identification was done through searching MS/MS data against the TMEW1 sequence with Mascot. The mass tolerance for the precursor and product ions were 7 ppm ad 0.02 Da, respectively.

10 μL of TMEW1 (5 μg) or 10 μL of TMEW1 & TMEB570 or TMEB675 mixture (5 μg) was incubated with 120 μL deuterium oxide labeling buffer (50 mM sodium phosphate, 100 mM sodium chloride at pH 7.4) for 0 s, 30 s, 360 s, 3600 s, or 14400 s at 10° C. Hydrogen/deuterium (H/D) exchange at each time point was performed in duplicate. Hydrogen/deuterium exchange was quenched by adding 130 μL of 4 M guanidine hydrochloride, 0.85 M TCEP buffer (final pH was 2.5). Subsequently, the quenched samples were subjected to on column pepsin/protease XIII digestion and LC-MS analysis as described above. The mass spectra were recorded in MS only mode.

Raw MS data was processed using HDX WorkBench, software for the analysis of H/D exchange MS data (Pascal et al., J. Am. Soc. Mass Spectrom. 2012, 23 (9), 1512-1521). The deuterium levels were calculated using the average mass difference between the deuteriated peptide and its native form (t0). About 97-99% of the protein could be mapped to specific peptides. The deuterium buildup curves showed significant difference in slopes, over exchange time for the peptides.

TMEB570 epitope: TMEW1 showed a modest reduction in deuterium uptake at residues 235-249 (residue numbering according to SEQ ID NO: 575), e.g. residues HGKCEHSINMQEPSC (SEQ ID NO: 592) within the membrane proximal region were protected from H/D exchange upon binding to TMEB570.

TMEB675 epitope: TMEW1 showed a modest reduction in deuterium uptake at residues 252-268 (residue numbering according to SEQ ID NO: 575), e.g. residues DAGYTGQHCEKKDYSVL (SEQ ID NO: 600) within the membrane proximal region were protected from H/D/exchange upon binding to TMEB675.

TMEB570 TMEFF2 epitope residues thus encompassed HGKCEHSINMQEPSC (SEQ ID NO: 592) and TMEB675 epitope residues encompassed DAGYTGQHCEKKDYSVL (SEQ ID NO: 600). Both antibodies bound TMEFF2 within the membrane proximal region.

7-6 Generation of Bispecific TMEFF2×CD3 Antibodies

Select monospecific anti-TMEFF2 and anti-CD3 antibodies (see Example 1-7) were expressed as IgG4/κ. F405L and R409K substitutions (EU numbering) were made into the anti-CD3 antibodies while the anti-TMEFF2 antibodies had wild-type IgG4. In addition to position 405 and 409 substitutions, the IgG4 mAbs were engineered to have S228P, F234A and L235A substitution.

The monospecific antibodies were expressed and purified using standard methods using a Protein A column (HiTrap MabSelect SuRe column). After elution, the pools were dialyzed into D-PBS, pH 7.2.

Bispecific TMEFF2×CD3 antibodies were generated by combining a monospecific TMEFF2 mAb and a monospecific CD3 mAb in in vitro Fab arm exchange as described in Int. Patent Publ. No. WO2011/131746. Briefly, at about 1-20 mg/ml at a molar ratio of 1:1 of each antibody in PBS, pH 7-7.4 and 75 mM 2-mercaptoethanolamine (2-MEA) was mixed together and incubated at 25-37° C. for 2-6 h, followed by removal of the 2-MEA via dialysis, diafiltration, tangential flow filtration and/or spinned cell filtration using standard methods.

The bispecific antibodies were further purified after the in vitro Fab-arm exchange using hydrophobic interaction chromatography to minimize residual parental anti-TMEFF2 and anti-CD3 antibodies using standard methods.

Table 73 and Table 74 show the generated bispecific antibodies.

TABLE 73

Parental

Bispecific
(TMEFF2 arm/

antibody
CD3 arm)
HCDR1
HCDR2
HCDR3
LCDR1
LCDR2
LCDR3

TMCB132
TMEB675
582
584
587
18
588
22

CD3B376
662
663
664
671
673
690

TMCB131
TMEB570
583
585
16
19
21
23

CD3B376
662
663
664
671
673
690

TMCB150
TMEB762
582
584
587
18
588
603

CD3B376
662
663
664
671
673
690

TABLE 74

Parental

Bispecific
(TMEFF2

antibody
arm/CD3 arm)
VH
VL
HC
LC

TMCB132
TMEB675
25
28
32
35

CD3B376
652
661
640
676

TMCB131
TMEB570
589
29
33
36

CD3B376
652
661
640
676

TMCB150
TMEB762
604
607
614
615

CD3B376
652
661
640
676

7-7 Characterization of Bispecific TMEFF2×CD3 Antibodies
Purity Analysis by Analytical Ultra Centrifugation (AUC)

Analytical ultracentrifugation (AUC) allows for the determination of the size, shape, state of aggregation, and reversible interaction of macromolecules in solution. Sedimentation velocity (SV) is an AUC technique that allows for a concentration gradient of a macromolecule to move to the outer radius of the sample holder (cell) as the centrifuge spins. This enables the determination of the sedimentation coefficient which is a factor of the size and shape of a molecule, and it unique to each molecule. Beckman Optima AUC instrument was used for this purpose. The samples were loaded into centrifuge cells equipped with 1.2 cm Beckman centerpieces (rated to 50K rpm) and quartz windows. The cells are assembled and torqued to 130 lbs. The centrifuge cells were placed into an An-50 (8 hole) or An-60 (4 hole) rotor and placed within the AUC chamber. The temperature of the AUC was equilibrated to 20.5° C. for at least one hour with the rotor in the chamber before initiating the run. Runs were performed at 40K rpm for mAb sample with scan count (250 scans), frequency of scan collection (90 seconds), data resolution (10 μM), wavelength at 280 nm. The data were analyzed using the direct boundary fitting software SEDANAL. Purity of the bispecific antibody TMCB150 and its parental mAbs were measured. TMCB150 showed 97.1% monomer, 2.8% dimer monomer and no aggregation, as determined by AUC meeting acceptable criteria for transiently expressed research material for further biophysical characterization. TMEB762 showed 95.5% monomer, 4.5% dimer and no aggregation while CD3B376 showed 97.7% monomer and 2.2% dimer with no aggregation. Aggregate levels of >5% of a minimum two-step purified molecule could have a significant impact on biological activity, solubility, stability and shelf-life.

Conformational Stability of Anti-TMEFF2/CD3 Bispecific by DSC

TMCB150 thermal unfolding was determined by DSC (Differential Scanning calorimetry) that showed an onset of unfolding Tm=52. 6° C., the first thermal transition (Tm1) at 61.8° C., second thermal transition (Tm2) at 67.6° C. and third thermal transition (Tm3) at 75.5° C. Based on parental antibodies (anti-TMEFF2, TMEB762 and anti-CD3, CD3B376) thermal transition profile as assessed by DSC before, Tm1 of TMCB150 corresponds to CD3B376 FAB unfolding and Tm3 of TMCB150 corresponds to TMEB762 Fab unfolding transitions.

Serum Stability

Serum stability assay is developed to evaluate properties of lead candidates for non-specific or off-target binding to human serum components. This may be predictive of poor pharmacokinetics and bio distribution properties. Binding and stability of TMCB150 is evaluated in both buffer and human serum using a fluorescence-based chromatography method. Bispecific antibody is labeled with Alexa Fluor 488 conjugate (Invitrogen kit according to manufacturer's instructions), incubated in Hepes buffer and human serum (Sigma, cat #H4522) at 4° C. and 37° C. for 2 days and then analysed by SEC-HPLC using Agilent HPLC system equipped with fluorescence detector. Percent aggregate is calculated from the integration of area under the curve of each peak. TMCB150 showed 2.4% aggregation in Hepes buffer at time zero and 2.0% and 1.3% aggregation after two days at 4° and 37° Celcius, respectively. In human serum, TMCB150 showed 1.7% aggregation at time zero and 1.1% aggregation after two days at both 4° and 37° Celcius.

Non-Specific Binding

Non-specific binding of the lead molecule to unrelated surfaces is determined by biosensor technology (Biacore 8K). Antibody is passed over SPR surfaces coated with unrelated proteins. If the antibody displays significant binding to these irrelevant surfaces, its predicted to have poor in-vivo properties and exhibit manufacturing challenges. Lead molecule is tested at the final concentration of 104. Irrelevant surfaces include negatively charged protein (Soy Trypsin Inhibitor), positively charged protein (Lysozyme and β-defensin), hydrophobic (Rh-integrin a4b7), human IgG, sticky protein (Rh-CD19). Proper controls are used in this experiment. Lead is flown over two surfaces, one is a blank and the other has a molecule directly immobilized. The response unit (RU) level is determined by subtracting the blank RU from the test surface RU. The RU of TMCB150 and the parental mAbs are given below in the table. A response unit ≥100 predicts high risks for non-specific binding to charged/hydrophobic/IgG surfaces which would create challenges during manufacturing and translate to poor PK properties. None of the antibodies show non-specific binding to irrelevant surfaces predicting low to no risks for manufacturing and in-vivo behavior.

TABLE 75

B-Defensin-
Human
Lyso-
rh-Integrin
rh-

STI
3
IgG
zyme
a4b7
CD19

TMCB150
1.6
6.4
7.5
4.2
0
3.4

TMEB762
0
4.1
8.4
4.6
0
3.3

CD3B376
19.5
3.1
0.4
0.2
0
0

High Concentration Stability

Many monoclonal antibodies are formulated at high concentration (≥100 mg/ml) to reduce injection volume to facilitate subcutaneous administration. In addition, all monoclonal antibodies are exposed to transiently high concentrations (≥50 mg/ml) during the purification process. High concentration stability is therefore a critical quality attribute for these molecules. Concentration is performed using centrifugal ultrafiltration devices with 30 kDa MWCO membranes. 5.1 to 5.3 mg of each protein were initially diluted to the same starting concentration and centrifuged at 4000×g in 15-minute intervals. At the end of each 15-minute centrifugation step, the concentrators are removed from the centrifuge and a visual estimate of the remaining sample volume is recorded. Concentration is measured by SoloVPE instrument. Concentrated samples were incubated at 4 C and 40C for 2 weeks and aliquots were drawn at timed intervals to check for the integrity by analytical SEC. TMCB150 and the parental mAbs (CD3B376 and TMEB762) concentrated normally. Final concentration of TMCB150 was 99.4 mg/mL and that of the parental mAbs were 52.2 mg/mL (TMEB762) and 52.6 mg/mL (CD3B376). Concentration remained the same for 2 weeks at 4 C and 40 C suggesting that molecules have good intrinsic properties with no potential for aggregation or adsorption to the eppendorf tubes. % species (A: Aggregate; M: Monomer; F: Fragment) as measured from SEC peak integration is provided below in the table. At high concentration, molecules are intact with 4-5% aggregates and ≤0.3% fragments after storing for 2 weeks at 40 C predicting good shelf life.

TABLE 76

Time Zero
2 weeks, 4° C.
2 weeks, 40° C.

A
M
F
A
M
F
A
M
F

TMCB150
0.8
99.2
0
1.4
98.6
0
3.6
96.1
0.3

TMEB762
2.5
97.5
0
2.7
97.3
0
3.1
96.9
0

CD3B376
1.1
98.9
0
2.5
97.5
0
5.1
94.7
0.2

Affinity Determination of Anti-TMEFF2/CD3 Bispecific Antibody by Kinetic Exclusion Assay (KinExA)

A KinExA 3200 instrument (Sapidyne Instruments, Inc.) was used to measure solution equilibrium affinity, K_D, of the bispecific mAb TMCB150 and its bivalent TMEFF2 parent TMEB762 mAb to human TMEFF2 extracellular domain (ECD). Serial dilutions of human TMEFF2 extracellular domain (ECD) were prepared with a constant concentration of anti-TMEFF2 mAb in 10 mM HEPES, 150 mM NaCL, 0.05% surfactant P20, pH 7.4, 0.1% BSA, and 0.02% NaN3. The reaction mixtures were incubated at RT until the binding interactions reached equilibrium. The duration of the incubation was determined using KinExA software simulation. Beads were prepared by direct covalent immobilization of TMEFF2-ECD by amine-coupling on pre-activated beads composed of bis-acrylamide/azlactone copolymer (Pierce Biotechnology, Inc.). After incubation the samples were run on the KinExA instrument to assess free antibody in the mixture by passing the mixture though the TMEFF2-modified beads, and detecting the captured antibody using a fluorescently labeled secondary antibody. The data was fit with a 1:1 binding model using the KinExA Pro software.

TABLE 77

mAb sample
K_DpM
95% CI, K_DpM

TMCB150
63.6
55.8-71.9

TMEB762
46.1
38.0-55.1

Measurement of Binding Affinity of Anti-TMEFF2/CD3 Bispecific Antibody to N-Terminal CD3ε Peptide

Kinetic rate constants were measured by SPR performed using Biacore 8K (GE Healthcare) and anti-human Fc biosensor surfaces. Anti-human immunoglobulin antibodies were covalently coupled to the surface of a CM4 sensor chip (GE Healthcare). Antibodies of interest were captured on the anti-human immunoglobulin sensor chip, followed by injection of N-terminal CD3ε peptide at various concentrations in HEPES Buffered Saline containing 0.05% surfactant P20 (Tween™ 20) and 100 ug/mL BSA. The surface was regenerated with 2 pulses injections of 30 μL of 0.8% phosphoric acid at 100 μL/min. Data reported is the difference in SPR signal between the flow cell containing the captured antibody and a reference cell without captured antibody. Additional instrumental contributions to the signal were removed by subtraction of the data from the blank injection from the reference-subtracted signal. The data were analyzed by fitting association and dissociation phases at all concentrations (global fit) with a 1:1 binding model using the Biaevaluation software (Biacore, Inc.). Data is reported as average+95% CI (Confidence Interval) which is calculated by t value for 95% CI*stdev/square root of the number of replicates.

TABLE 78

Average
Average
Average
95% CI,

mAb sample
k_on(1/Ms)
k_off(1/s)
K_D, nM
K_DpM

TMCB150
3.57E+04
1.03E−03
28.7
24.4-34.3

CD3B376
4.33E+04
1.17E−03
26.9
21.1-34.4

7-8 Bispecific TMEFF2×CD3 Antibodies are Effective in T-Cell Mediated Killing of Prostate Cancer Cells
T-Cell Mediated Killing of Prostate Cancer Cells

Select bispecific TMEFF2×CD3 antibodies were assessed for their ability to mediate T-cell mediated killing of prostate cancer cells.

T-cell mediated killing of the TMEFF2×CD3 bispecific antibodies was measured using a caspase cytotoxicity assay that indirectly measures cell killing via cleavage of a fluorescent substrate by active caspase 3/7. Cleavage of the substrate results in a fluorescent DNA dye, with fluorescence restricted to the cell nucleus. Repeated fluorescence measurements are taken in each well throughout the course of the assay, using a motorized 10× objective, capable of precisely imaging well(s) at the same coordinates. Target cell populations are identified based on defined size restrictions and/or through the use of a secondary label.

Frozen Pan CD3⁺ T-cells (Biological Specialty Corporation, Colmar, PA) were isolated by negative selection from normal healthy donors. Prostate cancer cells expressing TMEFF2 (LNCaP-AR) were cultured in RPMI 1640 with 10% HI FBS+supplements (Life Technologies). T-cells and target cells were combined at an effector to target ratio (E:T) of 3:1 in Phenol Red free RPMI+10% FBS and supplements (Life Technologies), without selection reagents, and 0.6 uL of NucView caspase reagent (Essen Bioscience) was added to each mL of cells, per manufacturer guidelines. A total volume of 0.1 mL cells were added to appropriate wells of a clear, 96-well flat-bottom plate (BD Falcon). TMEFF2×CD3 bispecific antibodies were prepared at 2× final concentration in Phenol Red free RPMI, prepared as indicated above, and 0.1 mL of compounds were added to each well. After 30 minute incubation at room temperature to minimize cell aggregation at the edge of wells, plates were transferred to the Zoom Incucyte instrument (Essen Bioscience) held at 37° C., 5% CO2.

After the assay was complete, each plate was analyzed using the appropriate processing definition. Raw fluorescent data was exported from the Incucyte Zoom software, and pasted into GraphPad Prism (GraphPad Software, Inc., La Jolla, CA). Caspase 3/7 activity was determined by calculating the area under the curve (AUC) for each well in GraphPad. AUC values were plotted as a function of Log 10 nM compound. The EC50 for each dose curve, in nanomolar (nM), was reported following non-linear regression analysis (4 parameter fit, least ordinary squares). Each assay contained a minimum of three biologic replicates, and each cell line was tested using T-cells from five healthy donors. Data were further analyzed by non-clinical statistics using a non-linear regression model.

Bispecific TMEFF2×CD3 Antibodies are Effective in In Vivo Prostate Cancer Tumor Models

Select bispecific antibodies were tested in ex vivo LnCaP prostate cancer model in male NSG mice. For the study, 10⁶LnCaP cells in 50% Cultrex in 0.2 mL/animal were administered by subcutaneous injection into right flank on Day 0. The animals were randomized when tumors reached a volume of about ˜100-150 mm³and injected 20⁶T-cells intraperitoneally/mouse at day 15. Ten animals were in each group. Treatment with the antibodies began 1-3 days post T-cell injections. Antibodies were administered intraperitoneally twice a week. Prior to dosing, all animals received IVIG+Fc Block. Tumor volume and body weight was assessed twice a week until tumors reached 1200 mm³. Treatment groups are shown in Table 79. CD3×Null antibody was used as a negative control in these assays, having a CD3 binding arm and a null arm binding HIV gp120.

TABLE 79

Group
Treatment
Dose

1
CD3 × Null
5
mg/kg

6
TMCB131 (TMEB570 × text missing or illegible when filed

5
mg/kg

7
TMCB131 (TMEB570 × text missing or illegible when filed

0.5
mg/kg

8
TMCB132 (TMEB675 × text missing or illegible when filed

5
mg/kg

9
TMCB132 (TMEB675 × text missing or illegible when filed

0.5
mg/kg

text missing or illegible when filed

indicates data missing or illegible when filed

Table 80 shows the percent tumor growth inhibition on day 38 per each group post tumor implant.

FIG. 71 shows the reduction in mean tumor volume over time post tumor implant. FIG. 72 shows the reduction in mean tumor volume of each mouse treated with 0.05, 0.1 or 0.5 mg/kg TMEB762×CD3B376.

TABLE 80

Treatment
% TGI on day 38

CNTO7008 (Null × CD3)

TMCB132 (TMEB675 × CD3B376) 5 mg/kg
48

TMCB132 (TMEB675 × CD3B376) 0.5 mg/kg
47.7

TMCB131 (TMEB570 × CD3B376) 5 mg/kg
23.3

TMCB131 (TMEB570 × CD3B376) 0.5 mg/kg
35

Efficacy of TMEB762×CD3B376 (TMCB150) was also evaluated in established LNCaP xenografts in male NOD.Cg-Prkdc^scidIl2rg^tm1Wjl/SzJ (NSG) mice humanized with 20e6 T cells. Animals were randomized into groups of 10 animals each by mean tumor volume on day 13 post tumor implantation. TMEB762×CD3B376 at 0.5, 0.1 and 0.05 mg/kg or null×CD3B376 antibody control at 0.5 mg/kg were dosed IP twice weekly for 5 weeks. On day 35 post-tumor implantation, when at least eight animals remained per group, tumor growth inhibition (% TGI) as determined by tumor volume was calculated. Statistically significant tumor growth inhibition was observed with TMEB762×CD3B376 at 0.5 and 0.1 mg/kg, but not at 0.05 mg/kg, as compared to null×CD3 control (FIG. 72, p≤0.0001). TMEB762×CDB376 at 0.5, 0.1, and 0.05 mg/kg elicited tumor growth inhibition of 110%, 88% and 25%, respectively, as compared to null×CD3 treated controls. TMEB762×CDB376 treatment resulted in seven and three complete responses at 0.5 and 0.1 mg/kg, respectively. Subcutaneous LNCaP tumors were measured by caliper twice weekly and the results presented as mean tumor volume (mm³)±SEM (>8 mice remaining per group).

T Cell Activation in Response to Administration of TMCB132

T cell activation in LnCaP prostate cancer cells was measured by flow cytometry, specifically by assessing CD25 positivity of CD3+/CD8+ T cells in 6 separate normal healthy donors (9642, 9672, 9762, 9772, 9832, 9852) 72 hours after treatment with TMCB132 (FIG. 73). Activation of CD3+ pan-T cells added at a 3:1 effector:target ratio was observed in response to administration of TMCB132 on LnCaP prostate cancer cells.

T-Cell Mediated Cytotoxicity of TMCB132 In Vitro.

T-cell-mediated cytotoxicity assay was used to evaluate the cytotoxicity potential of TMCB132 in vitro, using live-time lapse imaging on the Incucyte platform. TMCB132 was tested in TMEFF2 positive cell line LnCaP, in presence of isolated pan human CD3+ T cells from healthy donors at a (Effector:Target) effector:target ratio (E:T ratio) of 3:1. Cell death by apoptosis was monitored by measuring the fluorescence signal for active caspase-3/7 over a time period of 96 hours. TMCB132 promoted a dose-dependent reduction of viable LnCaP cells with increasing time. Dose-dependent increase in caspase-3/7 activity or fluorescence signal indicated cell death in LnCaP cells in presence of the T cells (FIG. 74). The data suggests that TMCB132 is effective in inducing T cell-mediated death in the LnCaP tumor cells.

Efficacy of TMCB132 in Established SC Human Prostate Xenograph in T-Cell Humanized Mice.

The antitumor efficacy of TMCB132 was evaluated in established subcutaneous (SC) human prostate LNCaP xenografts in male NOD.Cg-Prkdc^scidIl2rg^tm1Wjl/SzJ (NSG, The Jackson Laboratory, Bar Harbor, ME) mice humanized with 20^e6 T cells. Animals were randomized into groups of 10 animals each by mean tumor volume on day 13 post tumor implantation. TMCB132 at 0.5, 0.1 and 0.05 mg/kg or null×CD3B219 antibody control at 0.5 mg/kg were dosed IP twice weekly for 4 weeks. On day 45 post-tumor implantation, when at least seven animals remained per group, tumor growth inhibition (% TGI) as determined by tumor volume was calculated. Statistically significant tumor growth inhibition was observed with TMCB132 at 0.5 and 0.1 mg/kg and 0.05 mg/kg, as compared to null×CD3 control (FIG. 75, p≤0.0001). TMCB132 at 0.5, 0.1, and 0.05 mg/kg elicited tumor growth inhibition of 102%, 109% and 47%, respectively, as compared to null×CD3 treated controls. TMCB132 treatment resulted in three, two and one complete responses at 0.5, 0.1 and 0.05 mg/kg, respectively.

ANTI-CD3 ANTIBODIES AND USES THEREOF

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