BISPECIFIC ANTIBODIES BINDING TO CD7 AND CD33

Abstract

The present invention relates bispecific antibodies and antigen binding fragments thereof for binding to CD33 and CD7 for use in treating CD33+ CD7+ hematological malignancies, and in particular Acute Myeloid Leukaemia (AML). In particular, the present invention relates to a bispecific antibody or antigen binding fragments thereof binding to CD33 and CD7, wherein the bispecific antibody or antigen binding fragments comprises: a first binding region binding to human CD33 which has a K.sub.D binding affinity to CD33 of between about 1.36?10.sup.9 and about 9.23?10.sup.?8; and a second binding region binding to human CD7 which has a K.sub.D binding affinity to CD7 of between about 3.56?10.sup.?9 and about 5.41?10.sup.?7.

Claims

1. A bispecific antibody or antigen binding fragments thereof binding to CD33 and CD7, wherein the bispecific antibody or antigen binding fragments comprises: a first binding region binding to human CD33 which has a K.sub.D binding affinity to CD33 of between about 1.36?10.sup.?9 and about 9.23?10.sup.?8; and a second binding region binding to human CD7 which has a K.sub.D binding affinity to CD7 of between about 3.56?10.sup.?9 and about 2.29 ?10.sup.?7.

2. The antibody or antigen binding fragments thereof according to claim 1, wherein the first binding region binding to human CD33 has a K.sub.D binding affinity to CD33 of about 1.36?10.sup.?9 and the second binding region binding to human CD7 which has a K.sub.D binding affinity to CD7 of about 4.52 ?10.sup.?8.

3. The antibody or antigen binding fragments thereof according to claim 1, wherein the first binding region has a reduced K.sub.D binding affinity for CD33 compared to a wild type binding region that is able to bind to CD33.

4. The antibody or antigen binding fragments thereof according to claim 3, wherein the first binding region has a reduced K.sub.D binding affinity for CD33 of up to about 70 fold.

5. The antibody or antigen binding fragments thereof according to claim 4, wherein the first binding region has a reduced K.sub.D binding affinity for CD33 of up to about 68 fold.

6. The antibody or antigen binding fragments thereof according to claim 1, wherein the first binding region does not has a reduced K.sub.D binding affinity for CD33 compared to a wild type binding region.

7. The antibody or antigen binding fragments thereof according to claims 1 to 5, wherein the second binding region has a reduced binding affinity for CD7 compared to a wild type binding region that is able to bind to CD7.

8. The antibody or antigen binding fragments thereof according to claim 7, wherein the second binding region has a reduced K.sub.D binding affinity for CD7 of up to about 90 fold.

9. The antibody or antigen binding fragments thereof according to claim 8, wherein the second binding region has a reduced K.sub.D binding affinity for CD7 of up to about 87 fold.

10. The antibody or antigen binding fragments thereof according to claim 7, wherein the second binding region has a reduced K.sub.D binding affinity for CD7 in the range of about 9 to about 90 fold.

11. The antibody or antigen binding fragments thereof according to claim 10, wherein the second binding region has a reduced K.sub.D binding affinity for CD7 in the range of about 9 to about 87 fold.

12. The antibody or antigen binding fragments thereof according to claim 1, wherein both the first and/or second binding regions have a reduced binding affinity for their respective binding targets compared to respective wildtype binding regions ability to bind to the respective binding targets.

13. The antibody or antigen binding fragments thereof according to claim 12, wherein the first binding region has substantially the same K.sub.D binding affinity to CD33 when compared to a wild type binding region that is able to bind to CD33 and wherein the second binding region of has a reduced K.sub.D binding affinity for CD7 when compared to a wild type binding region that is able to bind to CD7.

14. The antibody or antigen binding fragments thereof according to claims 12 and 13 wherein the second binding region has a K.sub.D binding affinity for CD7 that is between about 9 and about 87 fold lower than that of a wild type binding region that is able to bind to CD7.

15. The antibody or antigen binding fragments thereof according to any preceding claim wherein: a) the first binding region has a K.sub.D binding affinity for CD33 that is selected from one of the following: about 1.36?10.sup.?9 or about 9.23?10.sup.?8; and/or b) the second binding region binding to human CD7 has a K.sub.D binding affinity to CD7 selected from one of the following: about 3.56?10.sup.?9, about 7.22?10.sup.?8, about 7.57?10.sup.?8, about 4.52?10.sup.?8, about 3.25?10.sup.?8 , about 5.41?10.sup.?7 or about 2.29?10.sup.?7.

16. The antibody or antigen binding fragments thereof according to claim 1, having a fold selectivity of CD7.sup.+/CD33.sup.+ vs CD7.sup.+/CD33.sup.? in the range of about 2 to about 20 and a fold selectivity of CD7.sup.+/CD33.sup.+ vs CD7.sup.?/CD33.sup.+ in the range of about 20 to about 70.

17. The antibody or antigen binding fragments thereof according to claim 16, having a fold selectivity of CD7.sup.+/CD33.sup.+ vs CD7.sup.+/CD33.sup.? in the range of about 5 to about 15 and a fold selectivity of CD7.sup.+/CD33.sup.+ vs CD7.sup.?/CD33.sup.+ in the range of about 20 to about 60.

18. The antibody or antigen binding fragments thereof according to claim 17, having a fold selectivity of CD7.sup.+/CD33.sup.+ vs CD7.sup.+/CD33.sup.? in the range of about 5 to about 15 and a fold selectivity of CD7.sup.+/CD33.sup.+ vs CD7.sup.?/CD33.sup.+ in the range of about 25 to about 55.

19. The antibody or antigen binding fragments thereof according to claim 18, having a fold selectivity of: a) CD7.sup.+/CD33.sup.+ vs CD7.sup.+/CD33.sup.? of about 14 and a fold selectivity of CD7.sup.+/CD33.sup.+ vs CD7.sup.?/CD33.sup.+ of about 48; b) CD7.sup.+/CD33.sup.+ vs CD7.sup.+/CD33.sup.? of about 6 and a fold selectivity of CD7.sup.+/CD33.sup.+ vs CD7.sup.?/CD33.sup.+ of about 48; or c) CD7.sup.+/CD33.sup.+ vs CD7.sup.+/CD33.sup.? of about 7 and a fold selectivity of CD7.sup.+/CD33.sup.+ vs CD7.sup.?/CD33.sup.+ of about 27.

20. The antibody or antigen binding fragments thereof according to claim 19, having a fold selectivity of: a) CD7.sup.+/CD33.sup.+ vs CD7.sup.+/CD33.sup.? of about 13.6 and a fold selectivity of CD7.sup.+/CD33.sup.+ vs CD7.sup.?/CD33.sup.+ of about 48.3; b) CD7.sup.+/CD33.sup.+ vs CD7.sup.+/CD33.sup.? of about 6.5 and a fold selectivity of CD7.sup.+/CD33.sup.+ vs CD7.sup.?/CD33.sup.+ of about 27.4; or c) CD7.sup.+/CD33.sup.+ vs CD7.sup.+/CD33.sup.? of about 6.1 and a fold selectivity of CD7.sup.+/CD33.sup.+ vs CD7.sup.?/CD33.sup.+ of about 47.5.

21. The antibody or antigen binding fragments thereof according to any one of claims 1 to 20 for use in the treatment of a CD7+CD33+ hematological malignancy.

22. The antibody or antigen binding fragments thereof for use according to claim 21, wherein said antibody or antigen binding fragments thereof is capable of inducing CD33 and/or CD7 receptor mediated internalization into a CD33+ and/or CD7+ cell.

23. The antibody or antigen binding fragments thereof for use according to claim 21 or 22, wherein the antibody or antigen binding fragments thereof bispecifically binds CD33 and CD7 and wherein the CD33+ and CD7+ cell is an AML cell.

24. The antibody or antigen binding fragments thereof for use according to any one of claims 21 to 23, wherein the antibody or antigen binding fragments thereof is capable of mediating antibody dependent cellular cytotoxicity.

25. The antibody or antigen binding fragments thereof for use according to any one of claims 21 to 23, wherein the antibody or antigen binding fragments thereof is attached to, or formed with an immune effector cell.

26. The antibody or antigen binding fragments thereof for use according to claim 25, wherein the immune effector cell comprises a T cell and/or a NK cell.

27. The antibody or antigen binding fragments thereof for use according to claim 26, wherein the cell inhibiting cell is a T cell.

28. The antibody or antigen binding fragments thereof for use according to claim 27, wherein the immune effector cell is a bispecific anti-CD33 anti-CD7 CAR-T.

29. The antibody or antigen binding fragments thereof for use according to claim 27, wherein the T cell comprises a CD33+ T cell, a CD7+ T cell, or a combination thereof.

30. The antibody or antigen binding fragments thereof for use according to any one of claims 25 to 26, wherein the antibody or antigen fragments thereof comprises: i) a cell killing portion; ii) a CD7 binding portion and iii) a CD33 binding portion.

31. The antibody or antigen binding fragments thereof for use according to claim 30, wherein said CD33 binding portion comprises an antigen binding fragments of an antibody.

32. The antibody or antigen binding fragments thereof for use according to claim 30 or claim 31, wherein said CD7 binding portion comprises an antigen binding fragments of an antibody.

33. The antibody or antigen binding fragments thereof for use according to any one of claims 30 to 32, wherein said cell killing portion is a cytotoxin.

34. The antibody or antigen binding fragments thereof for use according to claim 33, wherein said cytotoxin is selected from: i) a peptide toxin or ii) a chemical toxin.

35. The antibody or antigen binding fragments thereof for use according to any one of claims 30 to 32, further comprises a linking portion linking the cell kill portion with the CD7 binding portion and/or the CD33 binding portion.

36. The antibody or antigen binding fragments thereof for use according to any one of claims 30 to 34, in the format of an antibody drug conjugate.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0118] Embodiments of the invention are described below, by way of example only with reference to and as illustrated in the following figures:

[0119] FIG. 1 is a bar graph that shows the binding of affinity reduced bi-Fabs to CD33+/CD7+ cell lines, normal PBMC monocytes and T cells isolated from healthy human donors at 10 nM. 50000 cells of HNT-34 (CD33+/CD7+), Kasumi-3 (CD33+/CD7+), Jurkat (CD33-/CD7+), SHI-1 (CD33+/CD7?), healthy PBMC monocytes (CD33+/CD7?) and healthy T cells (CD33?/CD7+) were tested per CD33+/CD7+ bi-Fab antibody. The cells were resuspended in 100 ?l of 10 nM bi-Fab on ice for 1 hour. Following incubation, the cells were pelleted and incubated on ice with 50 ?l of Mouse anti-Human IgG Fab Secondary Antibody, PE diluted 1 in 17 in ice cold PBS/0.1% BSA for a further hour. Cells were washed and resuspended in PBS for analysis on the FACS Calibur. Data was plotted in excel;

[0120] FIG. 2 is a bar graph that shows the binding of each affinity reduced bi-Fab to CD33+/CD7+ cell lines, normal PBMC monocytes and T cells isolated from healthy human donors at 10 nM. 50000 cells of HNT-34 (CD33+/CD7+), Kasumi-3 (CD33+/CD7+), Jurkat (CD33?/CD7+), SHI-1 (CD33+/CD7?), healthy PBMC monocytes (CD33+/CD7?) and healthy T cells (CD33?/CD7+) were tested per CD33+/CD7+ bi-Fab antibody. The cells were resuspended in 100 ?l of 10 nM bi-Fab on ice for 1 hour. Following incubation, the cells were pelleted and incubated on ice with 50 ?l of Mouse anti-Human IgG Fab Secondary Antibody, PE diluted 1 in 17 in ice cold PBS/0.1% BSA for a further hour. Cells were washed and resuspended in PBS for analysis on the FACS Calibur. Data was plotted in excel;

[0121] FIG. 3 is a graph describing a cell kill assay conducted using a 10-point dose response of directly conjugated BVX130-MMAF on 2?10.sup.4 Kasumi-3 (CD33+/CD7+) cells per well. The plates were incubated at 37? C., 5% CO 2 for 96 hours. Following incubation, 5 ?l of Alamar Blue reagent was added per well and the plates read following a further incubation at 37? C., 5% CO.sub.2 for 4 and 6 hours. The data for each reading was plotted in GraphPad PRISM and the IC50 values recorded. The data from the 4-hour read for cell line Kasumi-3 was used to calculate the 1050. The IC50 was 0.1235 nM;

[0122] FIG. 4 is a graph describing a cell kill assay conducted using a 10-point dose response of directly conjugated BVX130-MMAF on 2?10.sup.4 HNT-34 (CD33+/CD7+) cells per well. The plates were incubated at 37? C., 5% CO 2 for 96 hours. Following incubation, 5 ?l of Alamar Blue reagent was added per well and the plates read following a further incubation at 37? C., 5% CO.sub.2 for 4 and 6 hours. The data for each reading was plotted in GraphPad PRISM and the IC50 values recorded. The data from the 4-hour read for cell line HNT-34s was used to calculate the 1050. 1050 was 0.1204 nM;

[0123] FIG. 5 is a graph describing a cell kill assay conducted using a 10-point dose response of directly conjugated BVX100-MMAF on 2?10.sup.4 Kasumi-3 (CD33+/CD7+) cells per well. The plates were incubated at 37? C., 5% CO.sub.2 for 96 hours. Following incubation, 5 ?l of Alamar Blue reagent was added per well and the plates read following a further incubation at 37? C., 5% CO.sub.2 for 4 and 6 hours. The data for each reading was plotted in GraphPad PRISM and the IC50 values recorded. The data from the 4-hour read for cell line Kasumi-3 was used to calculate the 1050. The IC50 was 0.0651 nM;

[0124] FIG. 6 is a graph describing a cell kill assay conducted using a 10-point dose response of directly conjugated BVX100-MMAF on 2?10.sup.4 HNT-34 (CD33+/CD7+) cells per well. The plates were incubated at 37? C., 5% CO.sub.2 for 96 hours. Following incubation, 5 ?l of Alamar Blue reagent was added per well and the plates read following a further incubation at 37? C., 5% CO.sub.2 for 4 and 6 hours. The data for each reading was plotted in GraphPad PRISM and the IC50 values recorded. The data from the 4-hour read for cell line HNT-34 was used to calculate the 1050. The IC50 was 0.2437 nM;

[0125] FIG. 7 is a graph that shows the dose response curve of a cell kill assay conducted using a 9-point dose of directly conjugated BVX130-MMAE or BVX100-MMAE on 2?10.sup.4 Jurkat cells (CD33?/CD7+) per well, to a top final concentration of 30 nM. The plates were incubated at 37? C., 5% CO2 for 96 hours. Following incubation, 10 ?l of CellTiter 96 AQueous One Solution was pipetted per well and the plates incubated at 37? C., 5% CO2 for a further 3 hours. The absorbance was read at 492 and 690 nm. The OD 690 nm was subtracted from the OD 492 nm and the data plotted using GraphPad PRISM software. Error bars represent the standard deviation of quadruplicate repeats;

[0126] FIG. 8 is a graph that shows the dose response curve of a cell kill assay conducted using a 10-point dose response of directly conjugated BVX130-MMAF or BVX100-MMAF on 2?10.sup.4 Jurkat cells (CD33?/CD7+) per well, to a top final concentration of 30 nM. The plates were incubated at 37? C., 5% CO2 for 96 hours. Following incubation, 5 ?l of Alamar Blue reagent was added per well and the plates read following a further incubation at 37? C., 5% CO2 for 4 and 6 hours. The data for each reading was plotted in GraphPad PRISM and the IC50 values recorded. The data from the 4-hour read for the Jurkat cell line was used to calculate the IC50. Error bars represent the standard deviation of quadruplicate repeats; and

[0127] FIG. 9 is a graph that shows the dose response curve of a cell kill assay conducted using a 10-point dose response of directly conjugated BVX130-MMAF or BVX100-MMAF on 2?10.sup.4 LOUCY cells (CD33?/CD7+) per well, to a top final concentration of 30 nM. The plates were incubated at 37? C., 5% CO2 for 96 hours. Following incubation, 5 ?l of Alamar Blue reagent was added per well and the plates read following a further incubation at 37? C., 5% CO2 for 4 and 6 hours. The data for each reading was plotted in GraphPad PRISM and the IC50 values recorded. The data from the 4-hour read for the LOUCY cell line was used to calculate the IC50. Error bars represent the standard deviation of quadruplicate repeats.

Example 1Assessing the Binding of Different CD33+/CD7+ bi-Fabs in Cell Lines and Primary Cells

[0128] The binding of different CD33+/CD7+ conjugated bispecific antibodies was assessed in a range of cell lines and the primary cells derived from healthy human donors. Each of the conjugated bispecific antibodies demonstrated a different pair of binding arms that were targeted towards either CD33 or CD7 (table 1). Each of the binding arms of the bispecific antibodies demonstrate different affinities for either CD33 or CD7.

TABLE-US-00001 TABLE 1 Bispecific antibodies assessed for binding to a range of cellular test system. Construct # Candidate # CD7 CD33 Mutant BVX0011/BVX1001 0 0 CD33-PP-1 WT/CD7-PP-1 WT BVX1101 1 0 CD33-PP-1 WT/CD7-PP-2 BVX1201 2 0 CD33-PP-1 WT/CD7-PP-3 BVX1301 3 0 CD33-PP-1 WT/CD7-PP-6 BVX1401 4 0 CD33-PP-1 WT/CD7-PP-7 BVX1501 5 0 CD33-PP-1 WT/CD7-PP-8 BVX1601 6 0 CD33-PP-1 WT/CD7-PP-13 BVX1011 0 1 CD33-PP-4/CD7-PP-1 WT BVX1611 6 1 CD33-PP-4/CD7-PP-13 BVX1021 0 2 CD33-PP-7/CD7-PP-1 WT BVX1521 5 2 CD33-PP-7/CD7-PP-8 BVX1621 6 2 CD33-PP-7/CD7-PP-13 BVX1631 6 3 CD33-PP-11/CD7-PP-13

TABLE-US-00002 TABLE 2 Nomenclature used to differentiate and identify each bispecific antibody. For example, BVX1301 corresponds to programme number 1, CD7 construct 3, CD33 construct WT, and MMAE payload. BVX1302 would replace the MMAE payload with MMAF. Nomenclature BVX Programme # CD7 CD33 Dash Payload # Construct # Construct # ()

Reagents

[0129]

TABLE-US-00003 Mouse anti-Human IgG Fab Secondary ThermoFisher #MA110377 Antibody, PE Jurkat Cells DSMZ 282 Kasumi-3 Cells DSMZ 714 HNT-34 Cells DSMZ 600 SHI-1 Cells DSMZ 645 Healthy PBMCs Donor 1 Cambridge Bioscience ID PR18E125592 PBS/A (Cat #. 50086470) VWR BSA (Bovine serum albumin ThermoFisher Fract V 100 ml 7.5% Cat #. 15260037) BVX1001 BVX1101 BVX1201 BVX1301 BVX1401 BVX1501 BVX1601 BVX1011 BVX1611 BVX1021 BVX1521 BVX1621 BVX1631

Method

[0130] 1 ml of each bi-Fab agent was prepared at 10 nM in PBS/0.1% BSA. The cells were harvested and counted so there were enough cells to test each bi-Fab with each bi-Fab dilution, plus a sample labelled with secondary antibody only, using 50,000 cells per test. For the PBMC sample 100,000 cells were used per test. The cells were pelleted at 1000 rpm for 5 minutes at 4? C. and resuspended in ice cold PBS. 100 ?l aliquots of each cell sample was pipetted across wells of a V-shape bottomed 96 well plate on ice. The cells were pelleted at 1000 rpm for 5 minutes at 4? C. and the supernatant aspirated. The cells were resuspended in 100 ?l of 10 nM bi-Fab. The plates were incubated on ice for 1 hour. 75 ?l of ice-cold PBS was added to each well and the cells pelleted at 1000 rpm for 5 minutes at 4? C. The supernatant was aspirated and the cells resuspended in 50 ?l of Mouse anti-Human IgG Fab Secondary Antibody, PE diluted 1 in 17 in ice cold PBS/0.1% BSA. The plates were incubated on ice for 1 hour. 100 ?l of ice-cold PBS was added per well and the cells pelleted at 1000 rpm for 5 minutes at 4? C. The cells were resuspended in 300 ?l of ice-cold PBS, transferred to FACS tube and fluorescence analysed using the FACS Calibur, detecting PE staining in FL2.

[0131] The monocyte populations in the PBMC sample were identified from the Side scatter/Forward scatter dot plot. The data was plotted in excel.

Results

[0132] The binding of each bispecific antibody (1 nM and 10 nM) to each cell line was assessed alongside the binding to monocytes and T-cells isolated from healthy human donors (FIG. 1 and FIG. 2). It is clear from the data that the specific CD7 and CD33 constructs used within each bispecific construct has an impact on the binding to double (CD33+/CD7+) vs single (CD33+/CD7? or CD33?/CD7+) antigen positive cell lines and primary cells isolated from healthy human donors.

TABLE-US-00004 TABLE 2 Bispecific constructs Fold Fold FACS re- Re- fold duction duction re- in in Affinity duction binding binding Final from com- affinity affinity Position Yeild cell Affinity pared com- com- Single/ Position of of Post capt. from to pared pared Double Mutation Mutation Purifi- exp. SPR FACS PP to WT to PP Mutant # Mutant #1 #2 cation (??/ml) (Kd) binding WT (Kd WT (Kd) 7-AA-WT WT 1 mg 23 2.63E?09 7-AA-1 Single Heavy Chain CDR3 1 mg 46 4.45E?08 17 Tyr108Ala 7-AA-2 Single Heavy Chain CDR3 1 mg NO 3.44E?06 1310 Tyr107Ala BINDING 7-AA-3 Single Heavy Chain CDR3 0.5 mg NO 1.92E?07 73 Tyr106Ala BINDING 7-AA-4 Single Heavy Chain CDR3 Failure NA NA Tyr103Ala 7-AA-5 Single Light Chain CDR3 Failure NA NA Trp94Ala 7-AA-6 Single Light Chain CDR3 Failure NA NA Trp94Leu 7-AA-7 Double Heavy Chain CDR3 Light Failure NA NA Tyr108Ala Chain CDR3 Trp94Ala 7-AA-8 Double Heavy Chain CDR3 Light Failure NA NA Tyr107Ala Chain CDR3 Trp94Ala 7-AA-9 Double Heavy Chain CDR3 Light Failure NA NA Tyr106Ala Chain CDR3 Trp94Ala 7-AA-10 Double Heavy Chain CDR3 Light Failure NA NA Tyr103Ala Chain CDR3 Trp94Ala 7-AA-11 Double Heavy Chain CDR3 Light Failure NA NA Tyr108Ala Chain CDR3 Trp94Leu 7-AA-12 Double Heavy Chain CDR3 Light Failure NA NA Tyr107Ala Chain CDR3 Trp94Leu 7-AA-13 Double Heavy Chain CDR3 Light Failure NA NA Tyr106Ala Chain CDR3 Trp94Leu 7-AA-14 Double Heavy Chain CDR3 Light Failure NA NA Tyr103Ala Chain CDR3 Trp94Leu 7-AA-15 Double Heavy Chain CDR1 Light Not started NA NA Tyr32Ala Chain CDR3 Trp94Leu 7-AA-16 Double Heavy Chain CDR2 Light Not started NA NA Asn52Ala Chain CDR3 Trp94Leu 7-PP-2 Single Heavy Chain 32.7 mg 7.57E?08 34.60 64.92 28.84 21.26 Arg57Gly 7-PP-3 Single Heavy Chain 84.6 mg 3.25E?08 13.04 24.47 12.38 9.13 Arg57Lys 7-PP-4 Single Heavy Chain 55.4 mg 1992.00 3737.34 Tyr102Thr 7-PP-5 Single Heavy Chain 80.2 mg 268.30 503.38 Leu 105Gly 7-PP-6 Single Heavy Chain 72.3 mg 4.52E?08 9.76 18.32 17.22 12.70 Asp104Ala 7-PP-7 Double Heavy Chain 6.2 mg 5.41E?07 267.30 501.50 206.10 151.97 Arg57Gly/ Tyr108Ala 7-PP-8 Double Heavy Chain 63.4 mg 2.29E?07 96.75 181.52 87.24 64.33 Arg57Lys/ Tyr108Ala 7-PP-9 Double Heavy Chain 78.8 mg 275.30 516.51 Tyr102Thr/ Tyr108Ala 7-PP-10 Double Heavy Chain 88.0 mg 334.90 628.33 Leu106Gly/ Tyr108Ala 7-PP-11 Double Heavy Chain 57.7 mg 2.46E?07 55.85 104.78 93.71 69.10 Asp104Ala/ Tyr108Ala 7-PP-12 Single Light 71.0 mg 5.46E?09 0.002 0.004 2.08 1.53 Chain Gln27Ala 7-PP-1 21.8 mg 3.56E?09 0.53 1.00 1.36 WT 7-PP-13 Heavy Chain CDR3 50.6 mg 7.22E?08 28.38 53.25 27.50 20.28 (mut_1 7- Tyr108Ala AA-1) A. Fold Reduction in binding affinity Position Position Affinity compared Single/ of of from FACS to 33- Double Mutation Mutation SPR binding PP-1 WT Mutant # Mutant #1 #2 (Kd) (nM) (Kd, nM) 33-AA- Wildtype 2.96E?09 WT (WT) 33-AA-1 Single Heavy Chain CDR1 2.62E?09 33-AA-2 Single Heavy Chain CDR2 NA 33-AA-3 Single Heavy Chain CDR2 3.49E?09 33-AA-4 Single Heavy Chain CDR3 NA 33-AA-5 Single Heavy Chain CDR3 8.82E?09 33-AA-6 Single Light Chain CDR1 NA 33-AA-7 Single Light Chain CDR3 8.68E?09 33-AA-8 Double Heavy Chain CDR2 Light Chain 7.89E?07 CDR3 33-AA-9 Double Heavy Chain CDR2 Light Chain 9.24E?09 CDR3 33-AA-10 Double Heavy Chain CDR3 Light Chain NA CDR3 33-AA-11 Double Heavy Chain CDR3 Light Chain 1.19E?08 CDR3 33-AA-12 Double Heavy Chain CDR2 Light Chain NA CDR1 33-AA-13 Double Heavy Chain CDR2 Light Chain NA CDR1 33-AA-14 Double Heavy Chain CDR3 Light Chain NA CDR1 33-PP-2 Single Light Chain 1.28E?09 0.16 0.94 33-PP-3 Single Heavy Chain 1.95E?09 0.19 1.43 33-PP-4 Single Heavy Chain 5.27E?08 321.10 38.75 33-PP-5 Double Heavy Chain Light Chain 1.75E?09 0.18 1.29 33-PP-6 Double Heavy Chain 1.72E?09 0.15 1.26 33-PP-7 Double Heavy Chain 9.23E?08 464.50 67.87 33-PP-8 Single Light Chain 2.01E?07 686.5 147.79 33-PP-9 Double Heavy Chain Light Chain 3.07E?07 1245.00 225.74 33-PP-10 Double Heavy Chain Light Chain 2.72E?06 147818.00 2000.00 33-PP-1 1.36E?09 0.16 WT B. A. CD7 constructs; B. CD33 constructs

[0133] FIGS. 1 and 2 show that the reduction in binding affinity of the CD7 binding arm of a bispecific antibody that also contains the wild type CD33 binding arm does not affect the binding affinity to CD7+/CD33+ cell lines. In a surprising finding, the binding of the bispecific constructs to CD33-/CD7+ cells were affected by the affinity of both the CD7 and the CD33 arm used within the construct. Table 2 above shows the results of the bispecific antibodies tested and this data is summarised in Table 3 which includes the fold reduction in binding affinity of each binding arm of each bispecific antibody.

TABLE-US-00005 TABLE 3 Overview of the binding affinity (Kd) of each bispecific antibody candidate compared to wildtype as measured by SPR Fold reduction Fold reduction in binding in binding Construct CD7 CD33 affinity affinity Name construct construct vs 7-PP-1 WT vs 33-PP-1 WT BVX100 7-PP-1 WT 33-PP-1 WT 0 0 BVX110 7-PP-2 33-PP-1 WT 21.3 0 BVX120 7-PP-3 33-PP-1 WT 9.1 0 BVX130 7-PP-6 33-PP-1 WT 12.7 0 BVX140 7-PP-7 33-PP-1 WT 152 0 BVX150 7-PP-8 33-PP-1 WT 64.3 0 BVX160 7-PP-13 33-PP-1 WT 20.28 0 BVX101 7-PP-1 WT 33-PP-4 0 38.8 BVX102 7-PP-1 WT 33-PP-7 0 68 BVX152 7-PP-8 33-PP-7 87 68 BVX161 7-PP-13 33-PP-4 20.28 39 BVX162 7-PP-13 33-PP-7 20.28 68

Example 2Assessing Cytotoxicity in CD7+CD33+ Immortalised Cell Lines

[0134] The cytotoxicity (cell kill) efficiency of CD33+/CD7+ bispecific antibody drug conjugates was assessed in multiple CD33+/CD7+ cell lines.

Test Agents

[0135] BVX100-MMAFCD33 monobinder?CD7 monobinder bi-Fab conjugated to mcMMAF (cytotoxic payload) [0136] BVX130-MMAFCD33 monobinder?CD7 monobinder bi-Fab conjugated to mcMMAF (cytotoxic payload) with partially optimised CD7 monobinder (i.e. different CD7 protein sequence compared to BVX100-MMAF) [0137] CD33 x CD33-MMAFCD33 monobinder?CD33 monobinder bi-Fab conjugated to mcMMAF (cytotoxic payload) [0138] Gemtuzumab-MMAFGemtuzumab is the commercial CD33 monospecific IgG antibody component of Pfizer's ADC Mylotarg?, conjugated to mcMMAF (cytotoxic payload) [0139] All ADC conjugates used the same payload, conjugation technology and same Drug:Antibody Ratio

Reagents

[0140]

TABLE-US-00006 Kasumi-3 Cells DSMZ 714 HNT-34 Cells DSMZ 600 BVX130-MMAF (P01-32, 10.5 uM) In House - Head of Chemistry BVX130-MMAE (193-27-3, 49 uM) In House - Head of Chemistry BVX100-MMAF (193-15, 13 uM) In House - Head of Chemistry BVX100-MMAE (193-28, 34 uM) In House - Head of Chemistry BSA (Bovine serum albumin Fract V ThermoFisher 100 ml 7.5% Cat #. 15260037) PBS/A (Cat #. 50086470) VWR Corning 384 Well Clear Flat Bottom SLS Polystyrene TC-Treated Microplates alamarBlue? Cell Viability Reagent ThermoFisher (DAL1025) RPMI-1640 medium 21875034 Gibco, Life Technologies GlutaMAX? Supplement 35050061 Gibco, Life Technologies Fetal Bovine Serum, Gibco, Life Technologies Heat inactivated 10500064 Penicillin-Streptomycin Gibco, Life Technologies (10,000 U/mL) 15140122

Method

[0141] Kasumi-3 and HNT-34 cell lines were harvested, counted and the volume required to seed 2000 cells per well across 100 wells in 40 ?l media per well was calculated for a 384-well plate. A 10-point dose response of BVX130-MMAE, BVX130-MMAF, BVX100-MMAE and BVX100-MMAF were prepared in Assay Media (RPMI, 10% FBS, 1% Glutamax, 1% Pen/Strep) at 5? the final concentration with a top final concentration of 30 nM. 10 ?l of each dose was pipetted across triplicate wells in a 384 well plate and a separate plate was prepared for each cell line tested. 500,000 cells from each cell line were pipetted into 5 ml of Assay Media and the cells pelleted. Each cell pellet was resuspended in 10 mls of Assay Media and 40 ?l pipetted into the indicated wells. 50 ?l of Assay Media was pipetted in the Blank control wells and 50 ?l of PBS was pipetted into all the spare wells and the plates incubated at 37? C., 5% CO.sub.2 for 4 or 7 days. Following 4 days incubation 5 ?l of Alamar Blue reagent was added per well and the plates read following a further incubation at 37? C., 5% CO.sub.2 for 4 and 6 hours. The data for each reading was plotted in GraphPad PRISM and the IC50 values recorded. The data from the 4-hour read for cell lines Kasumi-3 and HNT-34 was used in the IC50 summary table.

Results

[0142] Example dose response curves for each of these assays are presented in FIGS. 3 to 6. Briefly, each of the CD33+/CD7+ bispecific antibody drug conjugates showed robust cytotoxicity in different CD33+/CD7+ cell lines. Table 4 below shows an overview of the average cytotoxicity IC50 values for each bispecific antibody in each cell type across multiple experiments.

TABLE-US-00007 TABLE 4 Mean IC50's for CD33+CD7+ cell lines Kasumi-3 and HNT-34 across multiple experiments with CD7/CD33 bispecific antibody drug conjugates Kasumi-3 HNT-34 Mean IC50 when treated with 0.16 0.37 BVX130-MMAF (nM) Number of Experiments (n=) 13 12 Mean IC50 when treated with 0.45 0.40 BVX130-MMAE (nM) Number of Experiments (n=) 6 8 Mean IC50 when treated with 0.14 0.19 BVX100-MMAE (nM) Number of Experiments (n=) 9 8 Mean IC50 when treated with 0.11 0.41 BVX100-MMAF (nM) Number of Experiments (n=) 6 4

[0143] FIG. 3 is a graph that shows the cytotoxicity dose response curve produced when Kasumi-3 cells are exposed to increasing concentrations of BVX130-MMAF. Kasumi-3 cells are CD33+/CD7+ and BVX130-MMAF is an affinity reduced CD33+/CD7+ bispecific antibody conjugated to a cytotoxic payload. The example IC50 of BVX130-MMAF in Kasumi-3 cells is 0.1235 nM and the average IC50 across thirteen experiments is 0.16 nM. The average IC50 of the same bispecific antibody conjugate in HNT-34 cells (CD33+/CD7+) across twelve experiments is 0.37 nM. FIG. 4 is an example dose response curve that is produced when HNT-34s cells are exposed to increasing concentrations of BVX130-MMAF.

[0144] BVX130-MMAF comprises a partially optimised CD7 sequence compared with BVX100-MMAF. FIGS. 5 and 6 are example dose response curves produced using BVX100-MMAF as the conjugated bispecific antibody drug conjugate. As with BVX130-MMAF the cell types tested were Kasumi-3 and HNT-34; both are CD33+/CD7+. The average 1050 of BVX100-MMAF in Kasumi-3 cells is 0.11 nM across six experiments. The average IC50 of BVX100-MMAF in HNT-34 cells is 0.41 nM across four experiments.

[0145] The MMAF conjugated bispecific antibodies generally demonstrate a higher IC50 in each cell type as shown in Table 4. For example, an average IC50 of 0.16 nM for BVX130-MMAF in Kasumi-3 cells versus an IC50 of 0.45 nM for BVX130-MMAE in the same cell type. The same is true when considering BVX100-MMAF vs BVX100-MMAE in the same cell line.

SUMMARY

[0146] The results described above demonstrate that each of the bispecific CD33+/CD7+ antibodies conjugated to a cytotoxic payload cause effective cell death in two CD33+/CD7+ cells lines. The next step was to determine whether exposure to these bispecific antibody drug conjugates would cause off-target cytotoxicity in cells that express just CD33 or CD7 as single targets, representing single antigen positive healthy cell populations in the blood. This was assessed by determining fold selectivity in cell kill in CD33+/CD7+ cell lines vs CD33+/CD7? and CD33?/CD7+ cell lines using the bispecific antibody panel conjugated to a cytotoxic payload.

Example 3Assessing Selectivity for CD33+/CD7+ Over CD33+/CD7? and CD33?/CD7+ Cells

[0147] In an effort to determine whether certain bispecific antibodies conjugated to cytotoxic payloads preferentially targeted CD33+/CD7+ cells over CD33+/CD7? and CD33?/CD7+ cells, a panel of double and single antigen positive immortalised cell lines were exposed to a range of conjugated CD7+/CD33+ bispecific antibodies and the cytotoxicity measured.

Cytotoxic Activity of Bi-Fab-MMAE Conjugates in CD33 and CD7 Double and Single Positive Cell Lines

Reagents

[0148]

TABLE-US-00008 Kasumi-3 cells DSMZ 714 HNT-34 cells DSMZ 600 SHI-1 cells DSMZ 645 MV4.11 cells DSMZ 102 Jurkat Cells DSMZ 282 BVX100-MMAE In House - Head of Chemistry BVX110-MMAE BVX120-MMAE BVX130-MMAE BVX140-MMAE BVX150-MMAE BVX160-MMAE BSA (Bovine serum albumin Fract V ThermoFisher 100 ml 7.5% Cat #. 15260037) PBS/A (Cat #. 50086470) VWR Corning 384 Well Clear Flat Bottom SLS Polystyrene TC-Treated Microplates alamarBlue? Cell Viability Reagent ThermoFisher (DAL1025) RPMI-1640 medium 21875034 Gibco, Life Technologies GlutaMAX? Supplement 35050061 Gibco, Life Technologies Fetal Bovine Serum, Gibco, Life Technologies Heat inactivated 10500064 Penicillin-Streptomycin Gibco, Life Technologies (10,000 U/mL) 15140122

Method

[0149] Cells were harvested, counted and resuspended at 2?10.sup.4 cells per 90 ?l growth media. An 8-point dose response of the bi-Fab-MMAE was prepared in growth media at 5 times the final assay concentration, with a final top concentration of 30 nM. 10 ?l of bi-Fab-MMAE titration was pipetted into a 384-well plate and each concentration was tested across duplicate wells. 40 ?l of cells were pipetted into the wells and the plates incubated at 37? C., 5% CO2 for 96 hours. Following incubation, 5 ?l of MTS reagent was pipetted per well and the plates incubated at 37? C., 5% CO2 for a further 3 hours. The absorbance was read at 492 and 690 nm. The OD 690 nm was subtracted from the OD 492 nm and the data plotted using GraphPad PRISM software.

TABLE-US-00009 TABLE 5 Cytotoxic Activity of CD33+/CD7+ Bispecific-MMAE Conjugates in CD33 and CD7 Double and Single Positive Cell Lines Fold Fold Selec- Selec- tivity tivity of of EC.sub.50 CD7.sup.+/ CD7.sup.+/ EC.sub.50 (nM) (nM) EC.sub.50 (nM) EC.sub.50 (nM) CD33.sup.+ CD33.sup.+ Bi- CD7.sup.+/CD33.sup.+ CD7.sup.+/ CD7.sup.?/CD33.sup.+ CD7.sup.?/CD33.sup.? vs vs Fab- HNT- Kasumi- CD33.sup.? MV4. SHI- DND- CD7.sup.+/ CD7.sup.?/ MMAE 34 3 Jurkat 11 1 Ramos 39 CD33.sup.? CD33.sup.+ BVX100 0.19 0.14 0.92 10.77 2.26 NR NR 5.4 38 (+0.04) (+0.07) (+0.22) (+3.69) (+0.89) BVX110 0.61 0.82 4.52 26.97 11.39 NR NR 6.5 27.4 (+0.01) (+0.38) (+1.16) (+1.51) (+0.99) BVX120 0.23 0.26 1.52 16.52 7.21 NR NR 6.1 47.5 (+0.02) (+0.11) (+0.34) (+0.86) (+0.91) BVX130 0.4 0.45 5.83 24.07 17.48 NR NR 13.6 48.3 (+0.19) (+0.07) (+1.25) (+3.25) (+3.28) BVX140 17.23 24.91 22.80 NR NR NR NR 1.1 >1.4 (+4.28) (+3.94) (+5.0) BVX150 0.63 0.76 1.60 9.19 5.23 NR NR 2.3 10.3 (+0.17) (+0.20) (+0.54) (+0.17) (+0.29) BVX160 2.22 1.32 7.31 NR NR NR NR 4.2 >17 (+0.24) (+0.14) (+2.14)

Results

[0150] The determined cytotoxicity for each conjugated bispecific antibody tested across the full cell line panel is given in Table 5, along with the fold selectivity of each construct for double positive CD33+/CD7+ cell lines compared to either CD33+/CD7? or CD33?/CD7+ cell lines. From these results, BVX130 was selected for further analysis due to the potent cytotoxicity seen in CD33+/CD7+ cell lines and the increased fold selectivity seen vs CD33+/CD7? and CD33?/CD7+ cell lines.

Cytotoxic Activity of BVX100 and BVX130 MMAF Conjugates in CD7 Single Positive Cell Lines

Reagents

[0151]

TABLE-US-00010 Jurkat Cells DSMZ 282 LOUCY Cells DSMZ 394 BVX130-MMAF (P01-32, 10.5 uM) In House - Head of Chemistry BVX100-MMAF (193-15, 13 uM) In House - Head of Chemistry BSA (Bovine serum albumin ThermoFisher Fract V 100 ml 7.5% Cat #. 15260037) PBS/A (Cat #. 50086470) VWR Corning 384 Well Clear Flat Bottom Polystyrene TC-Treated Microplates SLS alamarBlue? Cell Viability Reagent ThermoFisher (DAL1025) RPMI-1640 medium 21875034 Gibco, Life Technologies GlutaMAX? Supplement 35050061 Gibco, Life Technologies Fetal Bovine Serum, Heat inactivated Gibco, Life Technologies 10500064 Penicillin-Streptomycin (10,000 U/mL) Gibco, Life Technologies 15140122

Method

[0152] Jurkat and LOUCY cell lines were harvested, counted and the volume required to seed 2000 cells per well across 100 wells in 40 ?l media per well was calculated for a 384-well plate. A 10-point dose response of BVX130-MMAF and BVX100-MMAF were prepared in Assay Media (RPMI, 10% FBS, 1% Glutamax, 1% Pen/Strep) at 5? the final concentration with a top final concentration of 30 nM. 10 ?l of each dose was pipetted across triplicate wells in a 384 well plate and a separate plate was prepared for each cell line tested. 500,000 cells from each cell line were pipetted into 5 ml of Assay Media and the cells pelleted. Each cell pellet was resuspended in 10 mls of Assay Media and 40 ?l pipetted into the indicated wells. 50 ?l of Assay Media was pipetted in the Blank control wells and 50 ?l of PBS was pipetted into all the spare wells and the plates incubated at 37? C., 5% CO2 for 4 days. Following 4 days incubation 5 ?l of Alamar Blue reagent was added per well and the plates read following a further incubation at 37? C., 5% CO2 for 4 and 6 hours. The data for each reading was plotted in GraphPad PRISM and the IC50 values recorded.

Results

[0153] The affinity modulated CD33+/CD7+ bispecific antibody BVX130 conjugated to either MMAF or MMAE cytotoxic payloads demonstrate reduced cytotoxicity in CD7 single antigen positive cells compared to BVX100, as can be seen in FIGS. 7 to 9.

[0154] As has been demonstrated, CD33+/CD7+ bispecific antibodies conjugated to a cytotoxic payload cause robust cytotoxicity in immortalised CD33+/CD7+ cell lines (FIGS. 3 to 6). Reduced cytotoxicity was seen in constructs with a reduced affinity CD7 binding arm together with a high affinity CD33 binding arm in CD33+/CD7? cell lines (Table 5) and cell lines that express CD33?/CD7+ (FIGS. 7 to 9). This is the case when two different cytotoxic payloads are conjugated to the bispecific antibody. Together, this suggests that CD33+/CD7+ bispecific antibodies conjugated to a cytotoxic payload could be used to cause robust cytotoxicity in CD33+/CD7+ cells while causing reduced cytotoxicity in CD33 and CD7 single antigen positive cells, such as monocytes (CD33+/CD7?) and T cells (CD33?/CD7+).

[0155] The BVX102, BVX152, BVX162, BVX110, BVX120 and BVX130 constructs all showed promise as therapeutics, and in particular BVX130 with BVX110 and BVX 120 showing similar activity.

[0156] The forgoing embodiments are not intended to limit the scope of the protection afforded by the claims, but rather to describe examples of how the invention may be put into practice.

TABLE-US-00011 SequenceListings CD7-PP-1-WTCD7bindingarmsequence: VH: (SEQIDNo.1) [00001] [00002] VHCDR1: (SEQIDNo.2) [00003] VHCDR2: (SEQIDNo.3) [00004] VHCDR3: (SEQIDNo.4) [00005] VL: (SEQIDNo.5) [00006] [00007] VLCDR1: (SEQIDNo.6) [00008] VLCDR2: (SEQIDNo.7) [00009] VLCDR3: (SEQIDNo.8) [00010] Heavy: (SEQIDNo.9) QVQLQESGAELVKPGASVKLSCKASGYTFTSYWMHWVKQRPGQGLEWIGKINPSNGRTNYNEKFKSKAT LTVDKSSSTAYMQLSSLTSEDSAVYYCARGGVYYDLYYYALDYWGQGTTVTVSSASTKGPSVFPLAPSS KSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYCCN VNHKPSNTKVDKKVEPKSCDKT Light: (SEQIDNo.10) DIELTQSPATLSVTPGDSVSLSCRASQSISNNLHWYQQKSHESPRLLIKSASQSISGIPSRESGSGSGT DFTLSINSVETEDFGMYFCQQSNSWPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNN FYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK SENRGEC CD7-PP-2CD7bindingarmsequence: VH: (SEQIDNo.11) [00011] [00012] VHCDR1: (SEQIDNo.12) [00013] VHCDR2: (SEQIDNo.13) [00014] VHCDR3: (SEQIDNo.14) [00015] VL: (SEQIDNo.15) [00016] [00017] VLCDR1: (SEQIDNo.16) [00018] VLCDR2: (SEQIDNo.17) [00019] VLCDR3: (SEQIDNo.18) [00020] Heavy: (SEQIDNo.19) QVQLQESGAELVKPGASVKLSCKASGYTFTSYWMHWVKQRPGQGLEWIGKINPSNGGTNYNEKFKSKAT LTVDKSSSTAYMQLSSLTSEDSAVYYCARGGVYYDLYYYALDYWGQGTTVTVSSASTKGPSVEPLAPSS KSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLOSSGLYSLSSVVTVPSSSLGTQTYCCN VNHKPSNTKVDKKVEPKSCDKT Light: (SEQIDNo.20) DIELTQSPATLSVTPGDSVSLSCRASQSISNNLHWYQQKSHESPRLLIKSASQSISGIPSRESGSGSGT DFTLSINSVETEDFGMYFCQQSNSWPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNN FYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK SENRGEC CD7-PP-3CD7bindingarmsequence: VH: (SEQIDNo.21) [00021] [00022] VHCDR1: (SEQIDNo.22) [00023] VHCDR2: (SEQIDNo.23) [00024] VHCDR3: (SEQIDNo.24) [00025] VL: (SEQIDNo.25) [00026] [00027] VLCDR1: (SEQIDNo.26) [00028] VLCDR2: (SEQIDNo.27) [00029] VLCDR3: (SEQIDNo.28) [00030] Heavy: (SEQIDNo.29) QVQLQESGAELVKPGASVKLSCKASGYTFTSYWMHWVKQRPGQGLEWIGKINPSNGKTNYNEKFKSKAT LTVDKSSSTAYMQLSSLTSEDSAVYYCARGGVYYDLYYYALDYWGQGTTVTVSSASTKGPSVFPLAPSS KSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYCCN VNHKPSNTKVDKKVEPKSCDKT Light: (SEQIDNo.30) DIELTQSPATLSVTPGDSVSLSCRASQSISNNLHWYQQKSHESPRLLIKSASQSISGIPSRESGSGSGT DFTLSINSVETEDFGMYFCQQSNSWPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNN FYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK SENRGEC CD7-PP-6bindingarmsequence: VH: (SEQIDNo.31) QVQLQESGAELVKPGASVKLSCKASGYTFTSYWMHWVKORPGQGLEWIGKINPSNGRTNYNEKFKSKAT LTVDKSSSTAYMQLSSLTSEDSAVYYCARGGVYYALYYYALDYWGQGTTVTVSS VHCDR1: (SEQIDNo.32) CKASGYTFTSYWMHWV VHCDR2: (SEQIDNo.33) WIGKINPSNGRTNYNEKFK VHCDR3: (SEQIDNo.34) CARGGVYYALY VL: (SEQIDNo.35) DIELTQSPATLSVTPGDSVSLSCRASQSISNNLHWYQQKSHESPRLLIKSASQSISGIPSRESGSGSGT DFTLSINSVETEDFGMYFCQQSNSWPYTFGGGTKLEIK VLCDR1: (SEQIDNo.36) [00031] VLCDR2: (SEQIDNo.37) [00032] VLCDR3: (SEQIDNo.38) [00033] Heavy: (SEQIDNo.39) QVQLQESGAELVKPGASVKLSCKASGYTFTSYWMHWVKQRPGQGLEWIGKINPSNGRTNYNEKFKSKAT LTVDKSSSTAYMQLSSLTSEDSAVYYCARGGVYYALYYYALDYWGQGTTVTVSSASTKGPSVFPLAPSS KSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYCCN VNHKPSNTKVDKKVEPKSCDKT Light: (SEQIDNo.40) DIELTQSPATLSVTPGDSVSLSCRASQSISNNLHWYQQKSHESPRLLIKSASQSISGIPSRESGSGSGT DFTLSINSVETEDFGMYFCQQSNSWPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNN FYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK SENRGEC CD7-PP-7CD7bindingarmsequence: VH: (SEQIDNo.41) [00034] [00035] VHCDR1: (SEQIDNo.42) [00036] VHCDR2: (SEQIDNo.43) [00037] VHCDR3: (SEQIDNo.44) [00038] VL: (SEQIDNo.45) [00039] [00040] VLCDR1: (SEQIDNo.46) [00041] VLCDR2: (SEQIDNo.47) [00042] VLCDR3: (SEQIDNo.48) [00043] Heavy: (SEQIDNo.49) QVQLQESGAELVKPGASVKLSCKASGYTFTSYWMHWVKQRPGQGLEWIGKINPSNGGTNYNEKFKSKAT LTVDKSSSTAYMQLSSLTSEDSAVYYCARGGVYYDLYYAALDYWGQGTTVTVSSASTKGPSVFPLAPSS KSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYCCN VNHKPSNTKVDKKVEPKSCDKT Light: (SEQIDNo.50) DIELTQSPATLSVTPGDSVSLSCRASQSISNNLHWYQQKSHESPRLLIKSASQSISGIPSRFSGSGSGT DFTLSINSVETEDFGMYFCQQSNSWPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNN FYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK SENRGEC CD7-PP-8CD7bindingarmsequence: VH: (SEQIDNo.51) [00044] [00045] VHCDR1: (SEQIDNo.52) [00046] VHCDR2: (SEQIDNo.53) [00047] VHCDR3: (SEQIDNo.54) [00048] VL: (SEQIDNo.55) [00049] [00050] VLCDR1: (SEQIDNo.56) [00051] VLCDR2: (SEQIDNo.57) [00052] VLCDR3: (SEQIDNo.58) [00053] Heavy: (SEQIDNo.59) QVQLQESGAELVKPGASVKLSCKASGYTFTSYWMHWVKQRPGQGLEWIGKINPSNGKTNYNEKFKSKAT LTVDKSSSTAYMQLSSLTSEDSAVYYCARGGVYYDLYYAALDYWGQGTTVTVSSASTKGPSVFPLAPSS KSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYCCN VNHKPSNTKVDKKVEPKSCDKT Light: (SEQIDNo.60) DIELTQSPATLSVTPGDSVSLSCRASQSISNNLHWYQQKSHESPRLLIKSASQSISGIPSRESGSGSGT DFTLSINSVETEDFGMYFCQQSNSWPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNN FYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK SENRGEC CD7-PP-12CD7bindingarmsequence: VH: (SEQIDNo.61) [00054] [00055] VHCDR1: (SEQIDNo.62) [00056] VHCDR2: (SEQIDNo.63) [00057] VHCDR3: (SEQIDNo.64) [00058] VL: (SEQIDNo.65) [00059] [00060] VLCDR1: (SEQIDNo.66) [00061] VLCDR2: (SEQIDNo.67) [00062] VLCDR3: (SEQIDNo.68) [00063] Heavy: (SEQIDNo.69) QVQLQESGAELVKPGASVKLSCKASGYTFTSYWMHWVKQRPGQGLEWIGKINPSNGRTNYNEKFKSKAT LTVDKSSSTAYMQLSSLTSEDSAVYYCARGGVYYDLYYYALDYWGQGTTVTVSSASTKGPSVFPLAPSS KSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYCCN VNHKPSNTKVDKKVEPKSCDKT Light: (SEQIDNo.70) DIELTQSPATLSVTPGDSVSLSCRASASISNNLHWYQQKSHESPRLLIKSASQSISGIPSRESGSGSGT DETLSINSVETEDFGMYFCQQSNSWPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNN FYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK SENRGEC CD7-PP-13CD7bindingarmsequence: VH: (SEQIDNo.71) [00064] [00065] VHCDR1: (SEQIDNo.72) [00066] VHCDR2: (SEQIDNo.73) [00067] VHCDR3: (SEQIDNo.74) [00068] VL: (SEQIDNo.75) [00069] [00070] VLCDR1: (SEQIDNo.76) [00071] VLCDR2: (SEQIDNo.77) [00072] VLCDR3: (SEQIDNo.78) [00073] Heavy: (SEQIDNo.79) QVQLQESGAELVKPGASVKLSCKASGYTFTSYWMHWVKQRPGQGLEWIGKINPSNGRTNYNEKFKSKAT LTVDKSSSTAYMQLSSLTSEDSAVYYCARGGVYYDLYYAALDYWGQGTTVTVSSASTKGPSVFPLAPSS KSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYCCN VNHKPSNTKVDKKVEPKSCDKT Light: (SEQIDNo.80) DIELTQSPATLSVTPGDSVSLSCRASQSISNNLHWYQQKSHESPRLLIKSASQSISGIPSRESGSGSGT DFTLSINSVETEDFGMYFCQQSNSWPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNN FYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK SENRGEC CD33-PP-1-WTbindingarmsequence: VH: (SEQIDNo.81) EVQLVQSGAEVKKPGSSVKVSCKASGYTITDSNIHWVRQAPGQSLEWIGYTYPYNGGTDYNQKEKNRAT LTVDNPTNTAYMELSSLRSEDTAFYYCVNGNPWLAYWGQGTLVTVSS VHCDR1: (SEQIDNo.82) [00074] VHCDR2: (SEQIDNo.83) [00075] VHCDR3: (SEQIDNo.84) [00076] VL: (SEQIDNo.85) DIQLTQSPSTLSASVGDRVTIICRASESLDNYGIRFLTWFQQKPGKAPKLLMYAASNQGSGVPSRFSGS GSGTEFTLTISSLQPDDFATYYCQQTKEVPWSFGQGTKLEIK VLCDR1: (SEQIDNo.86) [00077] VLCDR2: (SEQIDNo.87) [00078] VLCDR3: (SEQIDNo.88) [00079] CD33-PP-4bindingarmsequence: VH: (SEQIDNo.89) EVQLVQSGAEVKKPGSSVKVSCKASGYTITDSNIHWVRQAPGQSLEWIGYTYPYNGGTDYNQKEKNRAT LTVDNPTNTAYMELSSLRSEDTAFYYCVNGNPYLAYWGQGTLVTVSS VHCDR1: (SEQIDNo.90) [00080] VHCDR2: (SEQIDNo.91) [00081] VHCDR3: (SEQIDNo.92) [00082] VL: (SEQIDNo.93) DIQLTQSPSTLSASVGDRVTIICRASESLDNYGIRFLTWFQQKPGKAPKLLMYAASNQGSGVPSRESGS GSGTEFTLTISSLQPDDFATYYCQQTKEVPWSFGQGTKLEIK VLCDR1: (SEQIDNo.94) [00083] VLCDR2: (SEQIDNo.95) [00084] VLCDR3: (SEQIDNo.96) [00085] CD33-PP-7bindingarmsequence: VH: (SEQIDNo.97) EVQLVQSGAEVKKPGSSVKVSCKASGYTITDSNIHWVRQAPGQSLEWIGYIYPYNGGTDYNQKFKNRAT LTVDNPTNTAYMELSSLRSEDTAFYYCVNGNPYLAAWGQGTLVTVSS VHCDR1: (SEQIDNo.98) [00086] VHCDR2: (SEQIDNo.99) [00087] VHCDR3: (SEQIDNo.100) [00088] VL: (SEQIDNo.101) DIQLTQSPSTLSASVGDRVTIICRASESLDNYGIRFLTWFQQKPGKAPKLLMYAASNQGSGVPSRESGS GSGTEFTLTISSLQPDDFATYYCQQTKEVPWSFGQGTKLEIK VLCDR1: (SEQIDNo.102) [00089] VLCDR2: (SEQIDNo.103) [00090] VLCDR3: (SEQIDNo.104) [00091] OptionalSignalSequenceUpstreamofVHandVLDomains (SEQIDNo.105) METDTLLLWVLLLWVPGST