Bispecific antibodies

Abstract

The present invention relates to bispecific binding molecules comprising a first antigen binding domain that binds to CD7 and a second antigen binding domain that binds CD33. The invention also relates to methods of treatment and diagnosis using said bispecific bindng molecules.

Description

[0001] Bispecific Antibodies

[0002] Technical Field of Invention

[0003] The present invention relates to bispecific antigen binding molecules which target CD33 and CD7. The invention also relates to methods and uses of the bispecific binding molecules.

[0004] Background to the Invention

[0005] Acute Myelogenous Leukaemia or Acute Myeloid Leukaemia (AML) is a heterogeneous haematological malignancy involving the clonal expansion of myeloid blasts in the bone marrow and peripheral blood. AML represents >90% of cases of adult acute leukaemia and remains a largely aggressive disease with a fulminant clinical course. Despite advances in therapeutic regimens and the current understanding of successful Haematopoietic Stem Cell Transplantation (HSCT), the mortality rate for patients with AML is still high. Of all adults diagnosed with AML, on average only a quarter will live for 5 years or more. In people aged over 65 years, the outlook reduces further to a mere 12% surviving for more than 5 years (www.cancerresearchuk.org). This is substantially lowerthan many other high incidence cancers which have received much greater investment over the last few decades, such as Breast, Prostate and Bowel Cancer, where 5-year survival is now 87%, 85%, 59%, respectively (www.cancerresearchuk.org). This highlights the degree of unmet need in AML and the urgent drive for more investment into novel therapeutics.

[0006] Current guidelines recommend that intensive treatment of AML be comprised of induction chemotherapy (including an anthracycline in combination with cytarabine) followed by consolidation therapy once patients have reached clinical and haematological remission. The mainstay of induction therapy consists of the 7+3’ or similar ‘10+3’ regimen, which combines 7 or 10 days of continuous infusion cytarabine with 3 days of an anthracycline. It is generally offered to patients with an intermediate to favourable prognosis and a low risk of treatment related mortality. Unfortunately, minimal residual disease often persists in Complete Remission (CR), and relapse is almost inevitable if treatment is discontinued. Therefore, any patient with a favourable response to induction therapy should either receive a second induction regimen or go on to receive consolidation therapy to achieve lasting remission.

[0007] Anthracyclines and cytarabine belong to the antimetabolite class of potent cytotoxic drugs and are commonly used in chemotherapy regimens as they target cells with a high Proliferative Index (PI) by inhibiting DNA replication. Debilitating side effects are a common occurrence with these agents as they are not sufficiently selective for malignant cells and therefore exhibit substantial damage to healthy tissues which also carry a high PI. Common side effects include bone marrow suppression associated with a high risk of life-threatening infections, nausea and vomiting, hair loss, bruising and bleeding complications and tumour lysis syndrome (www.macmillan.org.uk). Allogenic HSCT, whilst currently the only option for a cure in this disease setting, is still considered a last resort for many disease indications due to the high risk of mortality and morbidity associated with this treatment, often with treatment-related mortality rates as high as 40%. This treatment option is reliant on a suitable donor being available, and of those that do find a donor and survive the procedure, 30-45% of these patients will go on to relapse, many requiring additional transplants.

[0008] AML represents a problematic disease to treat as many of the proposed tumour antigens available for targeted therapies are non-specific for the malignant cell population. Conventional therapeutic approaches indicated in AML aim to target a single antigen expressed on the surface of the leukaemic blasts. Whilst these therapeutics are deemed ‘targeted therapies’, the antigens that they target are also expressed at high levels on a range of healthy heamatopoietic cells, required for a healthy immune system. This degree of on-target off-tissue toxicity on healthy cells demonstrated by these therapies, substantially limits their utility in the disease setting. This often results in clinicians having no option but to administer suboptimal doses to patients and a substantial number of patients dying as a result of the therapy alone.

[0009] CD33 is an AML antigen that has been extensively investigated in this setting and is a well- established and validated AML target, which is still the antigen of choice for many existing and novel therapeutics. Whilst CD33 is a preferred target for many drug developers in AML due to the wide expression of this antigen on AML cells and the perceived reduced expression on earlier CD34+ haematopoietic stem cells, the CD33 antigen is also a prevalent myeloid marker in healthy cell populations. Therefore, as expected with any CD33 targeting treatment, therapies targeting this antigen also have many off-target effects on healthy cell populations, causing significant depletion of any myeloid cell harbouring the antigen.

[0010] CD7 is an antigen commonly associated with thymocytes and mature T cells and is believed to play an essential role in T cell interactions and T / B cell interactions in early lymphoid development. This antigen does not currently represent a commonly targeted antigen in therapeutic development, assumed to be due to the extensive expression of this antigen on healthy T cell populations. Since the late 1980s, it has been evident that certain AML cell populations express CD7, an otherwise lymphoid restricted antigen, in this disease specific manner.

[0011] The CD7+ subtype of AML is associated with increased leucocytosis, poor response to chemotherapy and poor overall and disease-free survival (Kahl, C., et al. (2001) CD7+ and CD56+ Acute Myelogenous Leukemia is a Distinct Biologic and Clinical Disease Entity. Haematology and Blood Transfusion, 40 1 12-119). Clinically, CD7+ AML patients are younger, more frequently males, have a higher incidence of central nervous system involvement and are often associated with less well differentiated subtypes of AML, further linked to poorer outcome (Tien, H. and Wang, C. (1998) CD7 Positive Hematopoietic Progenitors and Acute Myeloid Leukemia and other Minimally Differentiated Leukemia, Leukemia and Lymphoma, 3 93-98).

[0012] WO2019 / 102234, WO2022136887 and WO2022136888 discloses the dual targeting of cell inhibiting agents to the cell surface receptors CD7 and CD33 in the treatment of haematological malignancy.

[0013] It is an object of the present invention to provide improved therapies for haematological malignancies, and in particular AML. Accordingly, there remains a need for more effective therapies with improved specificity for treating haematological malignancy, including AML.

[0014] Summary of the Invention

[0015] The present inventor has developed binding agents which target both cells expressing CD33 and CD7 with excellent specificity. Surprisingly it has been shown herein that these bispecific binding molecules can advantageously target cells expressing CD7+CD33+ wherein CD33 is expressed on said cell at a low level.

[0016] The data provided herein demonstrates that the bispecific molecules of the invention have the potential to target tumour heterogeneity, where different populations of cancer cells within a patient can express target antigens at both, high and low levels. The term “tumour heterogeneity” refers to the differences in the tumour microenvironment high mutational burden, hypoxic conditions and abnormal vasculature, wherein several malignant subpopulations often exist within a single tumour mass (Dagogo-Jack., Nat Rev Clin Oncol. 2018 Feb;15(2):81-94.) Tumour heterogeneity currently represents a substantial hurdle for drug developers in the antibodybased therapeutic sector. The binding molecules of the invention have shown good efficacy in vitro against a panel of cell lines displaying the cancer-specific CD33+CD7+ target fingerprint. Herein the inventors use a CD7+CD33+ HNT-34 AML cell line, which was specifically selected based on its low expression levels of CD33. This level of CD33 expression is likely to be below the threshold for effective treatment with a de facto CD33-targeting monospecific ADC, such as the only currently approved and marketed ADC in AML - gemtuzumab ozogamicin (Mylotarg®).

[0017] An aspect of the invention relates to a bispecific binding molecule, comprising a first antigen binding domain that binds to CD7 and a second antigen binding domain that binds CD33, wherein said first antigen binding domain comprises ai) a heavy chain variable (VH) region comprising SEQ ID NO. 21 or a sequence comprising at least 90%, 92%, 95%, 97% or 99% sequence identity thereto, and aii) a light chain variable (VL) region comprising SEQ ID NO. 23 or a sequence comprising at least 80%, 85%, 90%, 92%, 95%, 97% or 99% sequence identity thereto, or bi) a heavy chain variable (VH) region comprising SEQ ID NO. 25 or a sequence comprising at least 90%, 92%, 95%, 97% or 99% sequence identity thereto, and bii) a light chain variable (VL) region comprising SEQ ID NO. 27 or a sequence comprising at least 80%, 85%, 90%, 92%, 95%, 97% or 99% sequence identity thereto, or ci) a heavy chain variable (VH) region comprising SEQ ID NO. 29 or a sequence comprising at least 90%, 92%, 95%, 97% or 99% sequence identity thereto, and cii) a light chain variable (VL) region comprising SEQ ID NO. 31 or a sequence comprising at least 80%, 85%, 90%, 92%, 95%, 97% or 99% sequence identity thereto, or di) a heavy chain variable (VH) region comprising SEQ ID NO. 33 or a sequence comprising at least 90%, 92%, 95%, 97% or 99% sequence identity thereto, and dii) a light chain variable (VL) region comprising SEQ ID NO. 35 or a sequence comprising at least 80%, 85%, 90%, 92%, 95%, 97% or 99% sequence identity thereto, or ei) a heavy chain variable (VH) region comprising SEQ ID NO. 37 or a sequence comprising at least 90%, 92%, 95%, 97% or 99% sequence identity thereto, and eii) a light chain variable (VL) region comprising SEQ ID NO. 39 or a sequence comprising at least 80%, 85%, 90%, 92%, 95%, 97% or 99% sequence identity thereto.

[0018] An aspect of the invention relates to a bispecific binding molecule, comprising a first antigen binding domain that binds to CD7 and a second antigen binding domain that binds CD33, wherein said first antigen binding domain comprises ai) a heavy chain variable (VH) region comprising SEQ ID NO. 21 or a sequence comprising at least 90%, 92%, 95%, 97% or 99% sequence identity thereto, and aii) a light chain variable (VL) region comprising SEQ ID NO. 23 or a sequence comprising at least 80%, 85%, 90%, 92%, 95%, 97% or 99% sequence identity thereto, or bi) a heavy chain variable (VH) region comprising SEQ ID NO. 25 or a sequence comprising at least 90%, 92%, 95%, 97% or 99% sequence identity thereto, and bii) a light chain variable (VL) region comprising SEQ ID NO. 27 or a sequence comprising at least 80%, 85%, 90%, 92%, 95%, 97% or 99% sequence identity thereto, or ci) a heavy chain variable (VH) region comprising SEQ ID NO. 29 or a sequence comprising at least 90%, 92%, 95%, 97% or 99% sequence identity thereto, and cii) a light chain variable (VL) region comprising SEQ ID NO. 31 or a sequence comprising at least 80%, 85%, 90%, 92%, 95%, 97% or 99% sequence identity thereto, or di) a heavy chain variable (VH) region comprising SEQ ID NO. 33 or a sequence comprising at least 90%, 92%, 95%, 97% or 99% sequence identity thereto, and dii) a light chain variable (VL) region comprising SEQ ID NO. 35 or a sequence comprising at least 80%, 85%, 90%, 92%, 95%, 97% or 99% sequence identity thereto, or ei) a heavy chain variable (VH) region comprising SEQ ID NO. 37 or a sequence comprising at least 90%, 92%, 95%, 97% or 99% sequence identity thereto, and eii) a light chain variable (VL) region comprising SEQ ID NO. 39 or a sequence comprising at least 80%, 85%, 90%, 92%, 95%, 97% or 99% sequence identity thereto. wherein the bispecific binding molecule is capable of targeting cells expressing CD7+ CD33+, wherein said cells express CD33 at a low level.

[0019] An aspect of the invention relates to an isolated nucleic acid molecule encoding the bispecific binding molecule of the invention.

[0020] An aspect of the invention relates to a vector encoding the isolated nucleic acid of the invention.

[0021] An aspect of the invention relates to a host cell comprising the nucleic acid of the invention, or the vector of the invention.

[0022] An aspect of the invention relates to an immunoconjugate comprising the bispecific binding molecule of the invention.

[0023] An aspect of the invention relates to a pharmaceutical composition comprising the bispecific binding molecule of the invention and optionally a pharmaceutically acceptable excipient, and / or adjuvant.

[0024] An aspect of the invention relates to bispecific binding molecule according to the invention for use in the treatment of a malignancy.

[0025] An aspect of the invention relates to a method of treating or preventing a malignancy comprising administering a therapeutically effective amount of the bispecific binding molecule of the invention.

[0026] An aspect of the invention relates to the use of the bispecific binding molecule of the invention for the manufacture of a medicament for the treatment of a malignancy.

[0027] An aspect of the invention relates to an in vitro, ex vivo or in vivo method of detecting CD33+ / CD7+ cells within a biological sample, comprising obtaining a biological sample contacting said biological sample with a bispecific binding molecule according to the invention.

[0028] An aspect of the invention relates to an in vitro, ex vivo or in vivo method of delivering a payload to CD33+ / CD7+ cells within a biological sample, comprising obtaining a biological sample contacting said biological sample with a bispecific binding molecule according to the invention.

[0029] An aspect of the invention relates to kit comprising the bispecific binding molecule of the invention and optionally instructions for use.

[0030] Figures

[0031] Figure 1. IC50 values comparing Bi-Fab and asymmetrical (antiCD33 ScFvx antiCD7 Fab) ADCs containing the same variable regions across modalities (A vs B).

[0032] Average IC50 values ±SEM (n>2, biological repeats, 2 technical replicates) from a cell kill assay conducted using a 9-point dose response of maleimidocaproyl monomethylauristatin F (mcMMAF) conjugated Bi-Fab (antiCD33 x antiCD7) (A) or asymmetrical (antiCD33 ScFv x antiCD7 Fab) (B) ADCs on target cells KASUMI-3 (CD33+HIGH / CD7+), HNT-34 (CD33+LOW / CD7+), and non-target cells JURKAT (CD337CD7+), MV4-11 and SHI-1 (CD33+ / CD7 ) and DND-39 (CD337CD7 ) cells.

[0033] Figure 2. CD33+ / CD7+cell selectivity of Bi-Fab (A) and asymmetrical (antiCD33 ScFv x antiCD7 Fab) (B) ADCs containing the same variable domains across modalities (A vs B).

[0034] Selectivity index of mcMMAF conjugated Bi-Fab (antiCD33 x antiCD7) (A) or asymmetrical (antiCD33 ScFv x antiCD7 Fab) (B) ADCs when comparing target cells CD33+HIGH / CD7+KASUMI-3 (top) and HNT-34 (CD33+LOW / CD7+) (bottom) cells to non-target single antigen expressing (CD33+ / CD7- or CD337CD7+) cells, SHI-1 , MV4-11 and JURKAT. A value of 1 indicates equitoxicity of the ADC, a value of <1 indicates no preferential cytotoxicity to double positive antigen expressing cells and a value of >1 indicates preferential cytotoxicity to double positive antigen expressing cells.

[0035] Figure 3. IC50 values of asymmetrical (antiCD33 Fab x antiCD7 ScFv) ADCs with different antiCD33 sequences.

[0036] Average IC50 values ±SEM (n>2, biological repeats, 2 technical replicates) from a cell kill assay conducted using a 9-point dose response of mcMMAF conjugated asymmetrical (antiCD33 Fab x antiCD7 ScFv) ADCs on target KASUMI-3 (CD33+HIGH / CD7+), HNT-34 (CD33+LOW / CD7+), versus non-target JURKAT (CD337CD7+), MV4-11 and SHI-1 (CD33+ / CD7 ) and non-target double antigen negative DND-39 (CD337CD7 ) cells, respectively.

[0037] Figure 4. CD33+ / CD7+cell selectivity of asymmetrical (antiCD33 Fab x antiCD7 ScFv) ADCs containing different antiCD33 sequences.

[0038] Selectivity index of mcMMAF conjugated asymmetrical (antiCD33 Fab x antiCD7 ScFv) ADCs when comparing target CD33+HIGH / CD7+KASUMI-3 (top) and CD33+LOW / CD7+HNT-34 (bottom) cells to non-target single antigen expressing (CD33+ / CD7_or CD337CD7+) cells, SHI-1 , MV4-11 and JURKAT. A value of 1 indicates equitoxicity of the ADC, a value of <1 indicates no preferential cytotoxicity to double positive antigen expressing cells and a value of >1 indicates preferential cytotoxicity to double positive antigen expressing cells. Figure 5. BVX01-a0032-AB4A and BVX01-a0059-AB4A ADC were dosed at 10 mg / kg, twice weekly, 10mg I kg once weekly and 3mg I kg twice weekly, respectively, via intravenous administration for 28 days. Cytarabine was administered intraperitoneally and dosed at 20 mg I kg once daily, 5 days on, 2 days off for 28 days, respectively. Group sizes for target cell HNT- 34 (CD33+LOW / CD7+) tumour bearing female SCID mice were 12 per group for vehicle and cytarabine and 9 per group for BVX test articles (all dose groups). Lead bispecific ADC clones with different antiCD7 variable domains showed good efficacy in vivo, leading to a highly statistically significant reduction in tumour volume compared to vehicle control at day 28 post dosing start (tumour regressions of 93% reported for BVX01-a0032-AB4A ADC and 89% reported for BVX01-a0059-AB4A ADC at 10mg I kg twice weekly at day 28 post dosing start; p=<0.001***).

[0039] Figure 6. Humanised mouse models treated with BVX01-a0010-AB4A Bi-Fab ADC showing significantly improved safety profile compared to Mylotarg®.

[0040] Examination of different cell populations in humanised mouse models at day 7 or 14 treated with BVX01-a0010-AB4A and Mylotarg® (Gemtuzumab Ozogamicin; GO) respectively, following single dose administration of test agent on day 0.

[0041] Figure 7. Sequence alignment of variable domains for the non-humanised, parental BVX01- a0010-AB4A Bi-Fab ADC and humanised BVX01-a0032-AB4A. Underlined are the CDRs using the IMGT (https: / / www.imgt.org / IMGTScientificChart / Numbering / IMGTIGVLsuperfamily.html) system. In bold are amino acid sequence differences between the parental and humanised sequence.

[0042] Figure 8. RP-HPLC spectra at 280 nm for BVX01-a0059; Top: Spectrum of Bi-Fab BVX01- a0059-0 with no payload conjugated and interchain disulfide bonds intact; Middle: Spectrum of Bi-Fab BVX01-a0059-0 after it has been reduced with TCEP for 2 hours; Bottom: Spectrum of payload (mcMMAF) conjugated Bi-Fab BVX01-a0059-AB4A taken at T=0 to provide a baseline for any observed payload deconjugation. HC - heavy chain; LC - light chain

[0043] Figure 9. Lead Bi-Fab ADC clones are not toxic to CD33+ myeloid cells ex vivo.

[0044] Effect of lead antiCD33 x antiCD7 Bi-Fab ADC clones on the differentiation of healthy CD34+ progenitor cells to myeloid CD33+ cells within a colony forming unit assay. (n=>2, apart from BVX01-a0090-AB4A, n=1) % colony counts relative to control cells-only wells.

[0045] Figure 10. Lead bispecific ADCs show minimal toxicity to CD7+ T-cells ex vivo.

[0046] Dose response curves of cell kill assay conducted using a 9-point dose response of antiCD33 x antiCD7 lead Bi-Fab ADC clones compared to CD7xCD7 homodimer ADC on CD7+ T-cells isolated from huPBMCs. Error bars represent the standard error of the mean of 2 biological repeats (n=2) performed in duplicate.

[0047] Figure 11. Lead bispecific ADCs show no off-target toxicity via macropinocytosis in HUVEC cells. Dose response curves of cell kill assay conducted using a 9-point dose response of antiCD33 x antiCD7 lead Bi-Fab ADCs on HUVEC cells. Error bars represent the standard error of the mean of 2 biological repeats (n=2) performed in duplicate.

[0048] Figure 12. Dose Response curves and IC50S of antiCD33 x antiCD7 lead bispecific ADCs with different linker payload combinations.

[0049] Dose response curves of cell kill assay conducted using a 9-point dose response of different bispecific ADCs with mcMMAF or vcMMAF as linker payloads. Error bars represent the standard error of the mean of 2 biological repeats (n=2) performed in duplicate. IC50 values presented in table format.

[0050] Figure 13. Percentage change in the number of CD45dimcells (blasts) in AML BM 4 patient sample.

[0051] % change in the number of CD45dim cells (blasts) gated by flow cytometry, after LTC for 4 weeks with pre-exposure to BVX01-a0032-AB4A bispecific ADC, BVX01-a0059-AB4A bispecific ADC or Cytarabine for 72h. Black dotted line represents 100% control where cells were untreated.

[0052] Figure 14. Fold cell selectivity between the IC50 (A) or IC90 (B) values obtained for BVX01- a0032-AB4A bispecific ADC when comparing potency against CD33+CD7+Kasumi-3 target human AML cancer cells in cell cytotoxicity assay (average from n=20 experiments, method described above) versus healthy human myeloid cells in the CFU-GM assay (average from n=3 experiments, method described above).

[0053] Figure 15. % Bodyweight change of animals in the in vivo studies comparing BVX132 and BVX159 and Cytarabine control. All agents were well tolerated with no impact on bodyweight (except TA-2 3m / kg one mouse taken off study on day 15 due to continued bodyweight loss).

[0054] Figure 16. ELISA of BVX01-a0032 and BVX01-a0010 bi-Fabs. Curve showing binding of BVX01-a0010 (BVX130) and BVX01-a0032 (BVX132) Bi-Fabs to human CD7 antigen.

[0055] Figure 17. Dose Response curves and IC50S of Bi-Fab format ADCs with different linker payloads. Dose response curves of cell kill assay conducted using a 9-point dose response of different Bi-Fab ADCs with CL2A-SN38, NAMPT inhibitor linker 1 , mc-bis-(PEG2-MMAF) (branched) or DBM-MMAF (bridged) as linker payloads. Error bars represent the standard error of the mean of 2 biological repeats (n=2) performed in duplicate. IC50 values presented in table format.

[0056] Figure 18. BVX01-a0032-AB4A showed efficacy in 4 CD33+ / CD7+AML PBMC patient samples. A) Flow cytometry plots showing initial percentage of CD33+ / CD7+dual antigen positive blasts in AML PBMC patient samples prior to ADC treatment. B) Dose response curves in the same patient samples (top and bottom data points relate to same patient sample), following treatment with BVX01-a0032-AB4A Bi-Fab ADC or isotype control.

[0057] Figure 19. BVX01-a0032-AB4A and BVX01-a0059-AB4A ADC were dosed at 10 mg / kg twice weekly, via intravenous administration for 28 days once tumour volumes had reached an average size of 0.7 cm3. Group sizes for target cell HNT-34 (CD33+LOW / CD7+) tumour bearing female SCID mice were 12 per group for vehicle and 6 or 5 per group for BVX test articles, respectively. Lead Bi-Fab ADC clones with different antiCD7 variable domains showed good efficacy in vivo, leading to a statistically significant tumour regression compared to vehicle control at day 28 post dosing start (97% for BVX01-a0032-AB4A ADC and 99% BVX01-a0059- AB4A ADC, both at 10mg / kg twice weekly; p<0.001*** one-tailed t test.).

[0058] Figure 20. Kaplan Meier Survival plot of BVX01-a0032-AB4A and BVX01-a0059-AB4A Bi-Fab ADCs in large tumour model. Survival of mice treated with either BVX01 -a0032-AB4A or BVX01 - a0059-AB4A at 10mg / kg twice weekly or vehicle control. Study ended on day 172 with 1 mouse in BVX01-a0032-AB4A dosed group and 2 mice BVX01-a0059-AB4A dosed group not having reached study condition end point. ** p<0.01 ; *** p<0.001 (compared to untreated control).

[0059] Figure 21. Kaplan-Meier Survival Plot of BVX01-a0032-AB4A and BVX01-a0059-AB4A Bi-Fab ADCs. Survival of mice after treatment with Cytarabine (20mg / kg 5d on 2d off), BVX01-a0032- AB4A and BVX01-a0059-AB4A Bi-Fab ADCs (3mg / kg x2 week, 10mg / kg Ix week or 10mg / kg 2x week) or vehicle control. * p<0.05; *** p<0.001 ; **** p<0.0001 (compared to untreated control),#p<0.05, compared to Cytarabine.

[0060] Detailed Description

[0061] The embodiments of the invention will now be described. In the following passages, different embodiments are described. In the following passages, different aspects of the invention are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.

[0062] Generally, nomenclatures used in connection with, and techniques of, cell and tissue culture, pathology, oncology, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art. The methods and techniques of the present disclosure are generally performed according to conventional methods well-known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. See, e.g., Green and Sambrook et al., Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012) Therapeutic Monoclonal Antibodies: From Bench to Clinic, Zhiqiang An (Editor), Wiley, (2009); and Antibody Engineering, 2nd Ed., Vols 1 and 2, Ontermann and Dubel, eds., Springer- Verlag, Heidelberg (2010).

[0063] Enzymatic reactions and purification techniques are performed according to manufacturer's specifications, as commonly accomplished in the art or as described herein. The nomenclatures used in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients. Suitable assays to measure the properties of the molecules disclosed herein are also described in the examples.

[0064] Binding Molecules

[0065] The present inventors have advantageously identified antigen binding molecules which have enhanced characteristics. Where the antibody sequences are used in a bispecific format, they show high cell selectivity for cells expressing CD7+CD33+. The term “cell selectivity” as used herein refers to the ability of a binding molecule to bind a specific target cell over a non-target cell, in the present invention the binding molecule demonstrate high selectivity for cells expressing CD7+CD33+ compared to non-target cells which do not express CD7+CD33+. In some embodiments the binding molecules of the invention may show higher selectivity for cells expressing CD7+CD33+ compared to cells expressing CD7+ or cells expressing CD33+. As demonstrated herein the antibody sequences can be used to target cells expressing CD7+CD33+ at varying levels, allowing more effective targeting of these cells. The data presented herein provides supportive evidence that the bispecific antigen binding molecules of the invention have the potential to target tumour heterogeneity, where different populations of cancer cells within a patient can express target antigens at both, high and low levels. Tumour heterogeneity currently represents a substantial hurdle for drug developers in the antibodybased therapeutic sector. Binding molecules of the invention show good efficacy in vitro against a panel of cell lines displaying the cancer-specific CD33+CD7+ target fingerprint. In particular the binding molecules demonstrate good efficacy against a CD7+CD33+ cell line (CD7+CD33+ HNT-34 AML cell line) specifically selected based on its low expression levels of CD33. This level of CD33 expression is likely to be below the threshold for effective treatment with a de facto CD33-targeting monospecific ADC, such as the only currently approved and marketed ADC in AML - gemtuzumab ozogamicin (Mylotarg®). As such the binding molecules according to the present invention provide advantages over the currently approved therapies.

[0066] The binding molecules of the present invention may comprise one or more of the following features i) greater selectivity for target CD33+ CD7+ cells compared to non-target cells ii) the ability to bind CD33+ CD7+ cells, wherein said cells express CD33 at a low level iii) the ability to bind CD33+ CD7+ cells, wherein said cells express CD33 at a high level iv) an IC50 for CD33+CD7+ cells in the range of 0.005 to 5 nM, v) greater selectivity for target CD33+ CD7+ cells, wherein CD33 is expressed at a low level, compared to non-target cells vi) greater selectivity for target CD33+ CD7+ cells, wherein CD33 is expressed at a high level, compared to non-target cells vii) the ability to reduce tumour volume in vivo viii) does not significantly effect the number of healthy CD33+ myeloid cells ix) does not significantly effect neutrophil levels x) show good serum stability, low pH stability and thermal melt protein stability xi) when used in an immunoconjugate form, demonstrates low payload deconjugation e.g. <10%. xii) when used in an immunoconjugate form, demonstrate good safety profile with no toxicity in healthy CD7+ cells or CD33+ myeloid progenitors xiii) do not induce macropinocytosis xiv) when used in an immunoconjugate form, demonstrate the ability to induce cell selective cell death xv) the ability to reduce the frequency of leukemic-LTC-initiating cells (L-LTC-IC) xvi) increased in vivo mean survival compared to vehicle control and / or standard therapy (e.g., Cytaribine).

[0067] The present invention relates to a bispecific binding molecule, comprising a first antigen binding domain that binds to CD7 and a second antigen binding domain that binds CD33, wherein said first antigen binding domain comprises ai) a heavy chain variable (VH) region comprising SEQ ID NO. 21 or a sequence comprising at least 90%, 92%, 95%, 97% or 99% sequence identity thereto, and aii) a light chain variable (VL) region comprising SEQ ID NO. 23 or a sequence comprising at least 80%, 85%, 90%, 92%, 95%, 97% or 99% sequence identity thereto, or bi) a heavy chain variable (VH) region comprising SEQ ID NO. 25 or a sequence comprising at least 90%, 92%, 95%, 97% or 99% sequence identity thereto, and bii) a light chain variable (VL) region comprising SEQ ID NO. 27 or a sequence comprising at least 80%, 85%, 90%, 92%, 95%, 97% or 99% sequence identity thereto, or ci) a heavy chain variable (VH) region comprising SEQ ID NO. 29 or a sequence comprising at least 90%, 92%, 95%, 97% or 99% sequence identity thereto, and cii) a light chain variable (VL) region comprising SEQ ID NO. 31 or a sequence comprising at least 80%, 85%, 90%, 92%, 95%, 97% or 99% sequence identity thereto, or di) a heavy chain variable (VH) region comprising SEQ ID NO. 33 or a sequence comprising at least 90%, 92%, 95%, 97% or 99% sequence identity thereto, and dii) a light chain variable (VL) region comprising SEQ ID NO. 35 or a sequence comprising at least 80%, 85%, 90%, 92%, 95%, 97% or 99% sequence identity thereto, or ei) a heavy chain variable (VH) region comprising SEQ ID NO. 37 or a sequence comprising at least 90%, 92%, 95%, 97% or 99% sequence identity thereto, and eii) a light chain variable (VL) region comprising SEQ ID NO. 39 or a sequence comprising at least 80%, 85%, 90%, 92%, 95%, 97% or 99% sequence identity thereto.

[0068] The present invention relates to a bispecific binding molecule, comprising a first antigen binding domain that binds to CD7 and a second antigen binding domain that binds CD33, wherein said first antigen binding domain comprises ai) a heavy chain variable (VH) region comprising SEQ ID NO. 21 or a sequence comprising at least 90%, 92%, 95%, 97% or 99% sequence identity thereto, and aii) a light chain variable (VL) region comprising SEQ ID NO. 23 or a sequence comprising at least 80%, 85%, 90%, 92%, 95%, 97% or 99% sequence identity thereto, or bi) a heavy chain variable (VH) region comprising SEQ ID NO. 25 or a sequence comprising at least 90%, 92%, 95%, 97% or 99% sequence identity thereto, and bii) a light chain variable (VL) region comprising SEQ ID NO. 27 or a sequence comprising at least 80%, 85%, 90%, 92%, 95%, 97% or 99% sequence identity thereto, or ci) a heavy chain variable (VH) region comprising SEQ ID NO. 29 or a sequence comprising at least 90%, 92%, 95%, 97% or 99% sequence identity thereto, and cii) a light chain variable (VL) region comprising SEQ ID NO. 31 or a sequence comprising at least 80%, 85%, 90%, 92%, 95%, 97% or 99% sequence identity thereto, or di) a heavy chain variable (VH) region comprising SEQ ID NO. 33 or a sequence comprising at least 90%, 92%, 95%, 97% or 99% sequence identity thereto, and dii) a light chain variable (VL) region comprising SEQ ID NO. 35 or a sequence comprising at least 80%, 85%, 90%, 92%, 95%, 97% or 99% sequence identity thereto, or ei) a heavy chain variable (VH) region comprising SEQ ID NO. 37 or a sequence comprising at least 90%, 92%, 95%, 97% or 99% sequence identity thereto, and eii) a light chain variable (VL) region comprising SEQ ID NO. 39 or a sequence comprising at least 80%, 85%, 90%, 92%, 95%, 97% or 99% sequence identity thereto. wherein the bispecific binding molecule is capable of targeting cells expressing CD7+CD33+, wherein said cells express CD33+ at a low level.

[0069] The bispecific binding molecule of the invention is capable of targeting cells expressing CD7+ CD33+, wherein said cells express CD33 at a low level. The bispecific binding molecule of the invention is also capable of targeting cells expressing CD7+ CD33+, wherein said cells express CD33 at a high level.

[0070] The level of expression of a cell surface receptor such as CD33 or CD7 may be determined using methods known to the skilled person. The level of expression may refer to the number of receptors present. The number of receptors present on a cell can be calculated by determining the specific antigen binding capacity of a cell, suitable methods are set out in the Examples in particular Example 1 cell profiling. In an embodiment the level of expression refers to the number of receptors present on a cell as determined by specific antigen binding capacity of a cell. The bispecific binding molecule may target cells expressing CD7+CD33+, wherein said cells express CD33 at a high level. A high level of expression of CD33 may refer to the number of receptors present on a cell as determined by specific antigen binding capacity of a cell. A high level of expression of CD33 may refer to a specific antigen binding capacity, provided in Antibody- Binding Capacity (ABC) units, of a cell in the range of 15,000 to 45,000, 16,000 to 45,000, 17,000 to 45,000, 18,000 to 45,000, 19,000 to 45,000, 20,000 to 45,000, 21 ,000 to 45,000, 22,000 to

[0071] 45,000, 23,000 to 45,000, 24,000 to 45,000, 25,000 to 45,000, 26,000 to 45,000, 27,000 to

[0072] 45,000, 28,000 to 45,000, 29,000 to 45,000, 30,000 to 45,000, 31 ,000 to 45,000, 32,000 to

[0073] 45000, 33,000 to 45,000, 34,000 to 45,000, 35,000 to 45,000, 15,000 to 40,000, 16,000 to

[0074] 40,000, 17,000 to 40,000, 18,000 to 40,000, 19,000 to 40,000, 20,000 to 40,000, 21 ,000 to

[0075] 40,000, 22,000 to 40,000, 23,000 to 40,000, 24,000 to 40,000, 25,000 to 40,000, 26,000 to

[0076] 40,000, 27,000 to 40,000, 28,000 to 40,000, 29,000 to 40,000, 30,000 to 40,000, 31 ,000 to

[0077] 40,000, 32,000 to 40,000, 33,000 to 40,000, 34,000 to 40,000, 35,000 to 40,000. A high level of expression of CD33 may refer to a number of receptors in the range of 15,000 to 45,000, 16,000 to 45,000, 17,000 to 45,000, 18,000 to 45,000, 19,000 to 45,000, 20,000 to 45,000, 21 ,000 to 45,000, 22,000 to 45,000, 23,000 to 45,000, 24,000 to 45,000, 25,000 to 45,000, 26,000 to

[0078] 45,000, 27,000 to 45,000, 28,000 to 45,000, 29,000 to 45,000, 30,000 to 45,000, 31 ,000 to

[0079] 45,000, 32,000 to 45000, 33,000 to 45,000, 34,000 to 45,000, 35,000 to 45,000, 15,000 to

[0080] 40,000, 16,000 to 40,000, 17,000 to 40,000, 18,000 to 40,000, 19,000 to 40,000, 20,000 to

[0081] 40,000, 21 ,000 to 40,000, 22,000 to 40,000, 23,000 to 40,000, 24,000 to 40,000, 25,000 to

[0082] 40,000, 26,000 to 40,000, 27,000 to 40,000, 28,000 to 40,000, 29,000 to 40,000, 30,000 to

[0083] 40,000, 31 ,000 to 40,000, 32,000 to 40,000, 33,000 to 40,000, 34,000 to 40,000, 35,000 to

[0084] 40,000.

[0085] The bispecific binding molecule may target cells expressing CD7+CD33+, wherein said cells express CD33 at a low level. A low level of expression of CD33 may refer to the number of receptors present on a cell as determined by specific antigen binding capacity, provided in Antibody-Binding Capacity (ABC) units, of said cell. A low level of expression of CD 33 may refer to a specific antigen binding capacity of a cell in the range of 1 ,000 to 15,000, 1 ,000 to 14,000, 1 ,000 to 13,000, 1 ,000 to 12,000, 1 ,000 to 11 ,000, 1 ,000 to 10,000, 1 ,000 to 9,000, 1 ,000 to 8,000, 1 ,000 to 7,000, 1 ,000 to 6,000, 1 ,000 to 5,000, 2,000 to 15,000, 2,000 to 14,000, 2,000 to 13,000, 2,000 to 12,000, 2,000 to 11 ,000, 2,000 to 10,000, 2,000 to 9,000, 2,000 to 8,000, 2,000 to 7,000, 2,000 to 6,000, 2,000 to 5,000, 3,000 to 15,000, 3,000 to 14,000, 3,000 to 13,000, 3,000 to 12,000, 3,000 to 11 ,000, 3,000 to 10,000, 3,000 to 9,000, 3,000 to 8,000, 3,000 to 7,000, 3,000 to 6,000, 3,000 to 5,000, 4,000 to 15,000, 4,000 to 14,000, 4,000 to 13,000, 4,000 to 12,000, 4,000 to 11 ,000, 4,000 to 10,000, 4,000 to 9,000, 4,000 to 8,000, 4,000 to 7,000, 4,000 to 6,000, 4,000 to 5,000. A low level of expression of CD33 may refer to a number of receptors in the range of 1 ,000 to 15,000, 1 ,000 to 14,000, 1 ,000 to 13,000, 1 ,000 to 12,000, 1 ,000 to 11 ,000, 1 ,000 to 10,000, 1 ,000 to 9,000, 1 ,000 to 8,000, 1 ,000 to 7,000, 1 ,000 to 6,000,

[0086] I ,000 to 5,000, 2,000 to 15,000, 2,000 to 14,000, 2,000 to 13,000, 2,000 to 12,000, 2,000 to

[0087] I I ,000, 2,000 to 10,000, 2,000 to 9,000, 2,000 to 8,000, 2,000 to 7,000, 2,000 to 6,000, 2,000 to 5,000, 3,000 to 15,000, 3,000 to 14,000, 3,000 to 13,000, 3,000 to 12,000, 3,000 to 1 1 ,000, 3,000 to 10,000, 3,000 to 9,000, 3,000 to 8,000, 3,000 to 7,000, 3,000 to 6,000, 3,000 to 5,000, 4,000 to 15,000, 4,000 to 14,000, 4,000 to 13,000, 4,000 to 12,000, 4,000 to 11 ,000, 4,000 to 10,000, 4,000 to 9,000, 4,000 to 8,000, 4,000 to 7,000, 4,000 to 6,000, 4,000 to 5,000.

[0088] The bispecific binding molecule may target cells expressing CD7+CD33+, wherein said cells express CD7 at a high level. A high level of expression of CD7 may refer to the number of receptors present on a cell as determined by specific antigen binding capacity, provided in Antibody-Binding Capacity (ABC) units, of a cell. A high level of expression of CD7 may refer to a specific antigen binding capacity of a cell in the range of 15,000 to 45,000, 16,000 to 45,000, 17,000 to 45,000, 18,000 to 45,000, 19,000 to 45,000, 20,000 to 45,000, 21 ,000 to 45,000,

[0089] 22,000 to 45,000, 23,000 to 45,000, 24,000 to 45,000, 25,000 to 45,000, 26,000 to 45,000,

[0090] 27,000 to 45,000, 28,000 to 45,000, 29,000 to 45,000, 30,000 to 45,000, 31 ,000 to 45,000,

[0091] 32,000 to 45000, 33,000 to 45,000, 34,000 to 45,000, 35,000 to 45,000, 15,000 to 40,000,

[0092] 16,000 to 40,000, 17,000 to 40,000, 18,000 to 40,000, 19,000 to 40,000, 20,000 to 40,000,

[0093] 21 ,000 to 40,000, 22,000 to 40,000, 23,000 to 40,000, 24,000 to 40,000, 25,000 to 40,000,

[0094] 26,000 to 40,000, 27,000 to 40,000, 28,000 to 40,000, 29,000 to 40,000, 30,000 to 40,000,

[0095] 31 ,000 to 40,000, 32,000 to 40,000, 33,000 to 40,000, 34,000 to 40,000, 35,000 to 40,000. A high level of expression of CD33 may refer to a number of receptors in the range of 15,000 to 45,000, 16,000 to 45,000, 17,000 to 45,000, 18,000 to 45,000, 19,000 to 45,000, 20,000 to

[0096] 45,000, 21 ,000 to 45,000, 22,000 to 45,000, 23,000 to 45,000, 24,000 to 45,000, 25,000 to

[0097] 45,000, 26,000 to 45,000, 27,000 to 45,000, 28,000 to 45,000, 29,000 to 45,000, 30,000 to

[0098] 45,000, 31 ,000 to 45,000, 32,000 to 45000, 33,000 to 45,000, 34,000 to 45,000, 35,000 to

[0099] 45,000, 15,000 to 40,000, 16,000 to 40,000, 17,000 to 40,000, 18,000 to 40,000, 19,000 to

[0100] 40,000, 20,000 to 40,000, 21 ,000 to 40,000, 22,000 to 40,000, 23,000 to 40,000, 24,000 to

[0101] 40,000, 25,000 to 40,000, 26,000 to 40,000, 27,000 to 40,000, 28,000 to 40,000, 29,000 to

[0102] 40,000, 30,000 to 40,000, 31 ,000 to 40,000, 32,000 to 40,000, 33,000 to 40,000, 34,000 to

[0103] 40,000, 35,000 to 40,000.

[0104] The bispecific binding molecule may target cells expressing CD7+CD33+, and wherein CD7 is expressed at a low level. A low level of expression of CD7 may refer to the number of receptors present on a cell as determined by specific antigen binding capacity, provided in Antibody- Binding Capacity (ABC) units, of a cell. A low level of expression of CD7 may refer to a specific antigen binding capacity of a cell in the range of 1 ,000 to 15,000, 1 ,000 to 14,000, 1 ,000 to 13,000, 1 ,000 to 12,000, 1 ,000 to 11 ,000, 1 ,000 to 10,000, 1 ,000 to 9,000, 1 ,000 to 8,000, 1 ,000 to 7,000, 1 ,000 to 6,000, 1 ,000 to 5,000, 2,000 to 15,000, 2,000 to 14,000, 2,000 to 13,000, 2,000 to 12,000, 2,000 to 11 ,000, 2,000 to 10,000, 2,000 to 9,000, 2,000 to 8,000, 2,000 to 7,000, 2,000 to 6,000, 2,000 to 5,000, 3,000 to 15,000, 3,000 to 14,000, 3,000 to 13,000, 3,000 to 12,000, 3,000 to 11 ,000, 3,000 to 10,000, 3,000 to 9,000, 3,000 to 8,000, 3,000 to 7,000, 3,000 to 6,000, 3,000 to 5,000, 4,000 to 15,000, 4,000 to 14,000, 4,000 to 13,000, 4,000 to 12,000, 4,000 to 1 1 ,000, 4,000 to 10,000, 4,000 to 9,000, 4,000 to 8,000, 4,000 to 7,000, 4,000 to 6,000, 4,000 to 5,000. A low level of expression of CD7 may refers to a number of receptors in the range of 1 ,000 to 15,000, 1 ,000 to 14,000, 1 ,000 to 13,000, 1 ,000 to 12,000, 1 ,000 to 11 ,000, 1 ,000 to 10,000, 1 ,000 to 9,000, 1 ,000 to 8,000, 1 ,000 to 7,000, 1 ,000 to 6,000, 1 ,000 to 5,000, 2,000 to 15,000, 2,000 to 14,000, 2,000 to 13,000, 2,000 to 12,000, 2,000 to 1 1 ,000, 2,000 to 10,000, 2,000 to 9,000, 2,000 to 8,000, 2,000 to 7,000, 2,000 to 6,000, 2,000 to 5,000, 3,000 to 15,000, 3,000 to 14,000, 3,000 to 13,000, 3,000 to 12,000, 3,000 to 11 ,000, 3,000 to 10,000, 3,000 to 9,000, 3,000 to 8,000, 3,000 to 7,000, 3,000 to 6,000, 3,000 to 5,000, 4,000 to 15,000, 4,000 to 14,000, 4,000 to 13,000, 4,000 to 12,000, 4,000 to 11 ,000, 4,000 to 10,000, 4,000 to 9,000, 4,000 to 8,000, 4,000 to 7,000, 4,000 to 6,000, 4,000 to 5,000.

[0105] As used herein, the terms "homology" or “identity” generally refers to the percentage of amino acid residues in a sequence that are identical with the residues of the reference polypeptide with which it is compared, after aligning the sequences and in some embodiments after introducing gaps, if necessary, to achieve the maximum percent homology, and in some embodiments not considering any conservative substitutions as part of the sequence identity. Thus, the percent homology between two amino acid sequences is equivalent to the percent identity between the two sequences. Neither N- or C-terminal extensions, tags or insertions shall be construed as reducing identity or homology. Methods and computer programs for the alignment are well known. The percentage identity between two amino acid sequences can be determined using well known mathematical algorithms. As used herein the term “sequence identity” generally refers to the percentage of amino acid residues in a sequence that are identical and / or comprise conservative amino acid substitutions, where the substituted amino acid has similar physicochemical properties. For example, where a positively charged amino acid has been replaced by a different positively charged amino acid. Percentage identity may be calculated using tools such as “EMBOSS Needle” which implements the Needleman-Wunch algorithm for the pairwise percent identities or similarities, amino acids may be considered similar if they have a positive score in the BLOSUM62 matrix. In an embodiment, where the term “sequence identity” is used herein it may be replaced by the term “sequence similarity”.

[0106] Sequence identity is commonly defined with reference to the algorithm GAP (Wisconsin GCG package, Accelerys Inc, San Diego USA). GAP uses the Needleman and Wunsch algorithm to align two complete sequences, maximising the number of matches and minimising the number of gaps. Generally, default parameters are used, with a gap creation penalty equalling 12 and a gap extension penalty equalling 4. Use of GAP may be preferred but other algorithms may be used, e.g. BLAST (which uses the method of Altschul et al. (1990) J. Mol. Biol. 215: 405-410), FASTA (which uses the method of Pearson and Lipman (1988) PNAS USA 85: 2444-2448), or the Smith-Waterman algorithm (Smith and Waterman (1981) J. Mol Biol. 147: 195-197), or the TBLASTN program, of Altschul et al. (1990) supra, generally employing default parameters. In particular, the psi-Blast algorithm (Altschul et al (1997) Nucl. Acids Res. 25 3389-3402) may be used. Sequence identity may be defined using the Bioedit, ClustalW algorithm. Alignments were performed using Snapgene and based on MUSCLE (Multiple Sequence Comparison by Log- Expectation) algorithms (Edgar (2004a) Nucleic Acids Res 32: 1792-7; Edgar (2004b) BMC Bioinformatics 5:113).

[0107] In some embodiments, the invention encompasses variants of the bispecific binding molecule of the invention. A variant of any of the above antigen-binding domains is a molecule having one or more amino acid substitutions, deletions, insertions or other modifications, and which retains a biological function of the antibody, that is binding to the target antigen and, thus, variant antibody or antigen-binding fragment thereof can be sequence engineered. Modifications may include one or more substitution, deletion or insertion of one or more codons encoding the antibody that results in a change in the amino acid sequence as compared with the native sequence of the antibody or antigen-binding fragment thereof.

[0108] Amino acid substitutions can be the result of replacing one amino acid with another amino acid having similar structural and / or chemical properties, such as the replacement of a leucine with a serine, i.e., conservative amino acid replacements. Substitutions, insertions, additions or deletions may optionally be in the range of about 1 to 25 or 1 to 50, for example 1 to 5, 1 to 10, 1 to 15, 1 to 20 amino acids, for example 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids.

[0109] In one embodiment, the substitution (e.g. 1 , 2, 3, 4 or 5 amino acid substitutions), insertion, addition or deletion is in HCDR1 , HCDR2 and or HCDR3. In one embodiment, the substitutions, insertion, addition or deletion is in LCDR1 , LCDR2 and or LCDR3. In one embodiment, the substitution, insertion, addition or deletion is in a heavy chain framework region or a light chain framework region. In one embodiment, the substitution (e.g. 1 , 2, 3, 4 or 5 amino acid substitutions), insertion, addition or deletion is with the framework region for example in HFR1 , HFR2 HFR3 and / or HFR4. In one embodiment, the substitutions, insertion, addition or deletion is in LFR1 , LFR2 LFR3 and / or LFR4.

[0110] The variation allowed may be determined by systematically making insertions, deletions or substitutions of amino acids in the sequence and testing the resulting variants for activity exhibited by the full-length or mature native sequence. In one embodiment, the modification is a conservative sequence modification. As used herein, the term "conservative sequence modifications" is intended to refer to amino acid modifications that do not significantly affect or alter the binding characteristics of the antibody containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into an antibody of the invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0111] Thus, one or more amino acid residues within the CDR regions described herein can be replaced with other amino acid residues from the same side chain family and the altered antibody can be tested for retained function (i.e. antigen binding) using the functional assays described herein.

[0112] Thus, these amino acid changes can typically be made without altering the biological activity, function, or other desired property of the polypeptide, such as its affinity or its specificity for antigen. In general, single amino acid substitutions in nonessential regions of a polypeptide do not substantially alter biological activity. Furthermore, substitutions of amino acids that are similar in structure or function are less likely to disrupt the polypeptides' biological activity. Abbreviations for the amino acid residues that comprise polypeptides and peptides described herein, and conservative substitutions for these amino acid residues are shown in Table 2 below.

[0113] Table. Amino Acid Residues and Examples of Conservative Amino Acid Substitutions

[0114] A skilled person will know that there are different ways to identify, obtain and optimise the antigen-binding molecules as described herein, including in vitro and in vivo expression libraries. This is further described in the examples. Optimisation techniques known in the art, such as display (e.g., ribosome and / or phage display) and I or mutagenesis (e.g., error-prone mutagenesis) can be used. The invention therefore also comprises sequence optimised variants of the antibodies described herein.

[0115] Thus sequence modifications of the sequences described herein may be made to remove sequence liabilities, such as deamidation, isomerization, oxidization, glycation, integrin Binding or acid labile or PyroE sites.

[0116] The first antigen binding domain may comprise a heavy chain variable (VH) region comprising SEQ ID NO. 21 or a sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity thereto.

[0117] The first antigen binding domain may comprise a light chain variable (VL) region comprising SEQ ID NO. 23 or a sequence comprising at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity thereto.

[0118] The first antigen binding domain may comprise a heavy chain variable (VH) region comprising SEQ ID NO. 25 or a sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity thereto.

[0119] The first antigen binding domain may comprise a light chain variable (VL) region comprising SEQ ID NO. 27 or a sequence comprising at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity thereto.

[0120] The first antigen binding domain may comprise a heavy chain variable (VH) region comprising SEQ ID NO. 29 or a sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity thereto.

[0121] The first antigen binding domain may comprise a light chain variable (VL) region comprising SEQ ID NO. 31 or a sequence comprising at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity thereto.

[0122] The first antigen binding domain may comprise a heavy chain variable (VH) region comprising SEQ ID NO. 33 or a sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity thereto.

[0123] The first antigen binding domain may comprise a light chain variable (VL) region comprising SEQ ID NO. 35 or a sequence comprising at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity thereto.

[0124] The first antigen binding domain may comprise a heavy chain variable (VH) region comprising SEQ ID NO. 37 or a sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity thereto.

[0125] The first antigen binding domain may comprise a light chain variable (VL) region comprising SEQ ID NO. 39 or a sequence comprising at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity thereto.

[0126] The first antigen binding domain may comprise a heavy chain variable (VH) region comprising SEQ ID NO. 21 , 25, 29, 33, or 37 or a sequence comprising at least 90%, 92%, 95%, 97% or 99% sequence identity thereto, wherein the CDR sequences are the same as SEQ ID NO.21 and the framework regions comprise amino acid differences. For example, the first antigen binding domain may comprise a heavy chain variable (VH) region comprising a HC CDR1 comprising SEQ ID NO. 11 , a HC CDR2 comprising SEQ ID NO. 12, a HC CDR3 comprising SEQ ID NO. 13, and a heavy chain variable (VH) region comprising SEQ ID NO. 21 or a sequence comprising at least 90%, 92%, 95%, 97% or 99% sequence identity thereto. The first antigen binding domain may comprise a light chain variable (VL) region comprising SEQ ID NO. 23, 27, 31 , 35, or 39 or a sequence comprising at least 80%, 85%, 90%, 92%, 95%, 97% or 99% sequence identity thereto, wherein the CDR sequence are the same as SEQ ID NO.23 and the framework regions comprise amino acid differences. For example, the first antigen binding domain may comprise a light chain variable (VL) region comprising a LC CDR1 comprising SEQ ID NO. 14, a LC CDR2 comprising SEQ ID NO. 15, a LC CDR3 comprising SEQ ID NO. 16 and a light chain variable (VL) region comprising SEQ ID NO. 23 or a sequence comprising at least 80%, 85%, 90%, 92%, 95%, 97% or 99% sequence identity thereto.

[0127] The bispecific binding molecule may comprise a first antigen binding domain comprising ai) a heavy chain variable (VH) region comprising SEQ ID NO. 21 or a sequence comprising between 1 and 19 amino acid differences thereto, and aii) a light chain variable (VL) region comprising SEQ ID NO. 23 or a sequence comprising between 1 and 21 amino acid differences thereto, or bi) a heavy chain variable (VH) region comprising SEQ ID NO. 25 or a sequence comprising between 1 and 19 amino acid differences thereto, and bii) a light chain variable (VL) region comprising SEQ ID NO. 27 or a sequence comprising between 1 and 21 amino acid differences thereto, or ci) a heavy chain variable (VH) region comprising SEQ ID NO. 29 or a sequence comprising between 1 and 19 amino acid differences thereto, and cii) a light chain variable (VL) region comprising SEQ ID NO. 31 or a sequence comprising between 1 and 21 amino acid differences thereto, or di) a heavy chain variable (VH) region comprising SEQ ID NO. 33 or a sequence comprising between 1 and 19 amino acid differences thereto, and dii) a light chain variable (VL) region comprising SEQ ID NO. 35 or a sequence comprising between 1 and 21 amino acid differences thereto, or ei) a heavy chain variable (VH) region comprising SEQ ID NO. 37 or a sequence comprising between 1 and 19 amino acid differences thereto, and eii) a light chain variable (VL) region comprising SEQ ID NO. 39 or a sequence comprising between 1 and 21 amino acid differences thereto.

[0128] The bispecific binding molecule may comprise a first antigen binding domain comprising ai) a heavy chain variable (VH) region comprising SEQ ID NO. 21 or a sequence comprising between 1 and 19 amino acid differences thereto, and aii) a light chain variable (VL) region comprising SEQ ID NO. 23 or a sequence comprising between 1 and 21 amino acid differences thereto, or bi) a heavy chain variable (VH) region comprising SEQ ID NO. 25 or a sequence comprising between 1 and 19 amino acid differences thereto, and bii) a light chain variable (VL) region comprising SEQ ID NO. 27 or a sequence comprising between 1 and 21 amino acid differences thereto, or ci) a heavy chain variable (VH) region comprising SEQ ID NO. 29 or a sequence comprising between 1 and 19 amino acid differences thereto, and cii) a light chain variable (VL) region comprising SEQ ID NO. 31 or a sequence comprising between 1 and 21 amino acid differences thereto, or di) a heavy chain variable (VH) region comprising SEQ ID NO. 33 or a sequence comprising between 1 and 19 amino acid differences thereto, and dii) a light chain variable (VL) region comprising SEQ ID NO. 35 or a sequence comprising between 1 and 21 amino acid differences thereto, or ei) a heavy chain variable (VH) region comprising SEQ ID NO. 37 or a sequence comprising between 1 and 19 amino acid differences thereto, and eii) a light chain variable (VL) region comprising SEQ ID NO. 39 or a sequence comprising between 1 and 21 amino acid differences thereto. wherein the bispecific antibody is capable of targeting cells expressing CD7+CD33+, wherein said cells express CD33+ at a low level.

[0129] The amino acid differences may be insertions substitutions or deletions. In one embodiment, the amino acid difference e.g., substitution insertion, addition or deletion is with the framework region for example in HFR1 , HFR2 HFR3 and / or HFR4. In one embodiment, the substitutions, insertion, addition or deletion is in LFR1 , LFR2 LFR3 and / or LFR4.

[0130] The first antigen binding domain may comprise a heavy chain variable (VH) region comprising SEQ ID NO. 21 , 25, 29, 33, or 37 or a sequence comprising between 1 and 19 amino acid differences thereto, wherein the CDR sequence are the same as SEQ ID NO.21 , 25, 29, 33, or 37 and the framework regions comprise amino acid differences. For example, the first antigen binding domain may comprise a heavy chain variable (VH) region comprising a HC CDR1 comprising SEQ ID NO. 11 , a HC CDR2 comprising SEQ ID NO. 12, a HC CDR3 comprising SEQ ID NO. 13, and a heavy chain variable (VH) region comprising SEQ ID NO. 21 or a sequence comprising between 1 and 19 amino acid differences thereto. The first antigen binding domain may comprise a heavy chain variable (VH) region comprising a HC CDR1 comprising SEQ ID NO. 11 , a HC CDR2 comprising SEQ ID NO. 12, a HC CDR3 comprising SEQ ID NO. 13, and a heavy chain variable (VH) region comprising SEQ ID NO. 21 , 25, 29, 33, or 37.

[0131] The first antigen binding domain may comprise a light chain variable (VL) region comprising SEQ ID NO. 23, 27, 31 , 35, or 39 or a sequence comprising between 1 and 21 amino acid differences thereto, wherein the CDR sequences are the same as SEQ ID NO. 23, 27, 31 , 35, or 39 and the framework regions comprise amino acid differences. For example, the first antigen binding domain may comprise a light chain variable (VL) region comprising a LC CDR1 comprising SEQ ID NO. 14, a LC CDR2 comprising SEQ ID NO. 15, a LC CDR3 comprising SEQ ID NO. 16 and a light chain variable (VL) region comprising SEQ ID NO. 23 or a sequence comprising between 1 and 21 amino acid differences thereto. The first antigen binding domain may comprise a light chain variable (VL) region comprising a LC CDR1 comprising SEQ ID NO. 14, a LC CDR2 comprising SEQ ID NO. 15, a LC CDR3 comprising SEQ ID NO. 16 and a light chain variable (VL) region comprising SEQ ID NO. 23, 27, 31 , 35, or 39.

[0132] The bispecific binding molecule map comprise a first antigen binding domain comprising i) a heavy chain variable (VH) region comprisinga HC CDR1 comprising SEQ ID NO. 11 , a HC CDR2 comprising SEQ ID NO. 12, a HC CDR3 comprising SEQ ID NO. 13, and a heavy chain variable (VH) region comprising SEQ ID NO. 21 or a sequence comprising between 1 and 19 amino acid differences thereto wherein the amino acid differences are present in one or more of the framework regions, and ii) light chain variable (VL) region comprising a LC CDR1 comprising SEQ ID NO. 14, a LC CDR2 comprising SEQ ID NO. 15, a LC CDR3 comprising SEQ ID NO. 16 and a light chain variable (VL) region comprising SEQ ID NO. 23 or a sequence comprising between 1 and 21 amino acid differences thereto, wherein the amino acid differences are present in one or more of the framework regions.

[0133] The heavy chain variable region of the first antigen binding domain may comprise SEQ ID NO. 21 or a sequence comprising between 1 and 19 amino acid differences thereto, wherein the amino acid differences are present in one or more of the framework regions. In an embodiment 1 to 10, 1 to 9, 1 to 8 or 1 to 7 amino acid differences, compared to SEQ ID NO. 21 , may be present in FR1 of the VH domain. In an embodiment 1 to 5, or 1 to 4 amino acid differences, compared to SEQ ID NO. 21 , may be present in FR2 of the VH domain. In an embodiment 1 to 10, 1 to 9, 1 to 8 or 1 to 7 amino acid differences, compared to SEQ ID NO. 21 , may be present in FR3 of the VH domain. In an embodiment 1 or 2 amino acid differences, compared to SEQ ID NO. 21 , may be present in FR4 of the VH domain.

[0134] The light chain variable region of the first antigen binding domain may comprise SEQ ID NO. 23 or a sequence comprising between 1 and 21 amino acid differences thereto, wherein the amino acid differences are present in one or more of the framework regions. In an embodiment 1 to 8, 1 to 7, 1 to 6, 1 to 5 or 1 to 4 amino acid differences, compared to SEQ ID NO. 23, may be present in FR1 of the VH domain. In an embodiment 1 to 4, 1 to 3, 1 to 2, or 1 amino acid differences, compared to SEQ ID NO. 23, may be present in FR2 of the VH domain. In an embodiment 1 to 15, 1 to 14, 1 to 13, 1 to 12, 1 to 11 , 1 to 10, 1 to 9, 1 to 8, 1 to 7 or 1 to 6 amino acid differences, compared to SEQ ID NO. 23, may be present in FR3 of the VH domain. In an embodiment 1 or 2 amino acid differences, compared to SEQ ID NO. 23, may be present in FR4 of the VH domain. The bispecific binding molecule map comprise a first antigen binding domain comprising i) a heavy chain variable (VH) region comprisinga HC CDR1 comprising SEQ ID NO. 11 , a HC CDR2 comprising SEQ ID NO. 12, a HC CDR3 comprising SEQ ID NO. 13, and a heavy chain variable (VH) region comprising SEQ ID NO. 21 or a sequence comprising between 1 and 19 amino acid differences thereto, and ii) light chain variable (VL) region comprising a LC CDR1 comprising SEQ ID NO. 14, a LC CDR2 comprising SEQ ID NO. 15, a LC CDR3 comprising SEQ ID NO. 16 and a light chain variable (VL) region comprising SEQ ID NO. 23 or a sequence comprising between 1 and 7 amino acid differences thereto.

[0135] The bispecific binding molecule may comprise a first antigen binding domain comprising i) a heavy chain variable (VH) region comprising a sequence selected from SEQ ID NO.

[0136] 21 , SEQ ID NO. 25, SEQ ID NO. 29, SEQ ID NO. 33, or SEQ ID NO. 37 and ii) a light chain variable (VL) region comprising a sequence selected from SEQ ID NO. 23, SEQ ID NO. 27, SEQ ID NO. 31 , SEQ ID NO. 35, or SEQ ID NO. 39 wherein the bispecific antibody is capable of targeting cells expressing CD7+CD33+, wherein said cells express CD33 at a low level.

[0137] The first antigen binding domain may comprise a heavy chain variable (VH) region comprising a sequence selected from SEQ ID NO. 21 or a sequence comprising at least 90%, 92%, 95%, 97% or 99% sequence identity thereto, SEQ ID NO. 25 or a sequence comprising at least 90%, 92%, 95%, 97% or 99% sequence identity thereto, SEQ ID NO. 29 or a sequence comprising at least 90%, 92%, 95%, 97% or 99% sequence identity thereto, SEQ ID NO. 33 or a sequence comprising at least 90%, 92%, 95%, 97% or 99% sequence identity thereto, or SEQ ID NO. 37 or a sequence comprising at least 90%, 92%, 95%, 97% or 99% sequence identity thereto.

[0138] The first antigen binding domain may comprise a heavy chain variable (VH) region comprising a sequence selected from SEQ ID NO. 21 or a sequence comprising between 1 and 19 amino acid differences thereto, SEQ ID NO. 25 or a sequence comprising between 1 and 19 amino acid differences thereto, SEQ ID NO. 29 or a sequence comprising between 1 and 19 amino acid differences thereto, SEQ ID NO. 33 or a sequence comprising between 1 and 19 amino acid differences thereto, or SEQ ID NO. 37 or a sequence comprising between 1 and 19 amino acid differences thereto. The number of amino acid differences within the VH region of the first antigen binding domain may be between 1 and 19 amino acid residue, between 1 and 18 amino acid residues, between 1 and 17 amino acid residues, between 1 and 16 amino acid residues, between 1 and 15 amino acid residues, between 1 and 14 amino acid residues, between 1 and 13 amino acid residues, between 1 and 12 amino acid residues, between 1 and 11 amino acid residues, between 1 and 10 amino acid residues, between 1 and 9 amino acid residues, between 1 and 8 amino acid residues, between 1 and 7 amino acid residues, between 1 and 6 amino acid residues, between 1 and 5 amino acid residues, between 1 and 4 amino acid residues, between 1 and 3 amino acid residues, or between 1 and 2 amino acid residues. In an embodiment the amino acid differences are present in the framework region, for example in HFR1 , HFR2 HFR3 and / or HFR4. In an embodiment the amino acid differences are present in the framework region, for example in LFR1 , LFR2 LFR3 and / or LFR4.

[0139] The first antigen binding domain may comprise a light chain variable (VL) region comprising a sequence selected from SEQ ID NO. 23, or a sequence comprising at least 80%, 85%, 90%, 92%, 95%, 97% or 99% sequence identity thereto, SEQ ID NO. 27, or a sequence comprising at least 80%, 85%, 90%, 92%, 95%, 97% or 99% sequence identity thereto, SEQ ID NO. 31 , or a sequence comprising at least 80%, 85%, 90%, 92%, 95%, 97% or 99% sequence identity thereto, SEQ ID NO. 35, or a sequence comprising at least 80%, 85%, 90%, 92%, 95%, 97% or 99% sequence identity thereto, or SEQ ID NO. 39 or a sequence comprising at least 80%, 85%, 90%, 92%, 95%, 97% or 99% sequence identity thereto.

[0140] The first antigen binding domain may comprise a light chain variable (VL) region comprising a sequence selected from SEQ ID NO. 23, or a sequence comprising between 1 and 21 amino acid differences thereto, SEQ ID NO. 27, or a sequence comprising between 1 and 21 amino acid differences thereto, SEQ ID NO. 31 , or a sequence comprising between 1 and 21 amino acid differences thereto, SEQ ID NO. 35, or a sequence comprising between 1 and 21 amino acid differences thereto, or SEQ ID NO. 39 or a sequence comprising between 1 and 21 amino acid differences thereto. The number of amino acid differences within the VL region of the first antigen binding domain may be between 1 and 21 amino acid residues, between 1 and 20 amino acid residues, between 1 and 19 amino acid residues, between 1 and 18 amino acid residues, between 1 and 17 amino acid residues, between 1 and 16 amino acid residues, between 1 and 15 amino acid residues, between 1 and 14 amino acid residues, between 1 and 13 amino acid residues, between 1 and 12 amino acid residues, between 1 and 11 amino acid residues, between 1 and 10 amino acid residues, between 1 and 9 amino acid residues, between 1 and 8 amino acid residues, between 1 and 7 amino acid residues, between 1 and 6 amino acid residues, between 1 and 5 amino acid residues, between 1 and 4 amino acid residues, between 1 and 3 amino acid residues, or between 1 and 2 amino acid residues. In an embodiment the amino acid differences are present in the framework region, for example in HFR1 , HFR2 HFR3 and / or HFR4. In an embodiment the amino acid differences are present in the framework region, for example in LFR1 , LFR2 LFR3 and / or LFR4.

[0141] The bispecific binding molecule may comprise a first antigen binding domain comprising i) a heavy chain comprising SEQ ID NO. 22 or a sequence comprising at least 70%, 80%, 85%, 90%, 95% or 99% sequence identity thereto, and ii) a light chain comprising SEQ ID NO. 24 or a sequence comprising at least 70%, 80%, 85%, 90%, 95% or 99% sequence identity thereto wherein the bispecific antibody is capable of targeting cells expressing CD7+CD33+, wherein said cells express CD33 at a low level.

[0142] The bispecific binding molecule may comprise a first antigen binding domain comprising i) a heavy chain comprising a sequence selected from SEQ ID NO. 22, SEQ ID NO. 26, SEQ ID NO. 30, SEQ ID NO. 34, or SEQ ID NO. 38 and ii) a light chain variable (VL) region comprising a sequence selected from SEQ ID NO. 24, SEQ ID NO. 28, SEQ ID NO. 32, SEQ ID NO. 36, or SEQ ID NO. 40 wherein the bispecific antibody is capable of targeting cells expressing CD7+CD33+, wherein said cells express CD33 at a low level.

[0143] The first antigen binding domain may comprise a heavy chain comprising a sequence selected from SEQ ID NO. 22 or a sequence comprising at least 70%, 80%, 85%, 90%, 95% or 99% sequence identity thereto, SEQ ID NO. 26 or a sequence comprising at least 70%, 80%, 85%, 90%, 95% or 99% sequence identity thereto, SEQ ID NO. 30 or a sequence comprising at least 70%, 80%, 85%, 90%, 95% or 99% sequence identity thereto, SEQ ID NO. 34 or a sequence comprising at least 70%, 80%, 85%, 90%, 95% or 99% sequence identity thereto, or SEQ ID NO. 38 or a sequence comprising at least 70%, 80%, 85%, 90%, 95% or 99% sequence identity thereto.

[0144] The first antigen binding domain may comprise a light chain variable (VL) region comprising a sequence selected from SEQ ID NO. 24 or a sequence comprising at least 70%, 80%, 85%, 90%, 95% or 99% sequence identity thereto, SEQ ID NO. 28 or a sequence comprising at least 70%, 80%, 85%, 90%, 95% or 99% sequence identity thereto, SEQ ID NO. 32 or a sequence comprising at least 70%, 80%, 85%, 90%, 95% or 99% sequence identity thereto, SEQ ID NO. 36 or a sequence comprising at least 70%, 80%, 85%, 90%, 95% or 99% sequence identity thereto, or SEQ ID NO. 40 or a sequence comprising at least 70%, 80%, 85%, 90%, 95% or 99% sequence identity thereto.

[0145] The bispecific binding molecule may comprise a second antigen binding domain comprising i) a heavy chain variable (VH) region comprising a HC CDR1 comprising SEQ ID NO. 2, a HC CDR2 comprising SEQ ID NO. 3, a HC CDR3 comprising SEQ ID NO. 4, and ii) a light chain variable (VL) region comprising a LC CDR1 comprising SEQ ID NO. 7, a LC CDR2 comprising SEQ ID NO. 8, a LC CDR3 comprising SEQ ID NO. 9 wherein the bispecific binding molecule is capable of targeting cells expressing CD7+CD33+, wherein said cells express CD33+ at a low level.

[0146] The bispecific binding molecule may comprise a second antigen binding domain comprising i) a heavy chain variable (VH) region comprising SEQ ID NO. 1 and ii) a light chain variable (VL) region comprising SEQ ID NO. 6 wherein the bispecific antibody is capable of targeting cells expressing CD7+CD33+, wherein said cells express CD33+ at a low level.

[0147] An aspect of the invention relates to a bispecific binding molecule comprising a first antigen binding domain that binds to CD7 and a second antigen binding domain that binds CD33, wherein said first antigen binding domain comprising i) a heavy chain variable (VH) region comprising SEQ ID NO. 21 or a sequence comprising at least 70%, 80%, 85%, 90%, 95% or 99% sequence identity thereto, and ii) a light chain variable (VL) region comprising SEQ ID NO. 23 or a sequence comprising at least 70%, 80%, 85%, 90%, 95% or 99% sequence identity thereto.

[0148] The first antigen binding domain may comprise i) a heavy chain variable (VH) region comprising SEQ ID NO. 21 or a sequence comprising between 1 and 19 amino acid differences thereto, and ii) a light chain variable (VL) region comprising SEQ ID NO. 23 or a sequence comprising between 1 and 21 amino acid differences thereto.

[0149] The first antigen binding domain may comprise i) a heavy chain variable (VH) region comprising a sequence selected from SEQ ID NO.

[0150] 21 , SEQ ID NO. 25, SEQ ID NO. 29, SEQ ID NO. 33, or SEQ ID NO. 37 and ii) a light chain variable (VL) region comprising a sequence selected from SEQ ID NO. 23, SEQ ID NO. 27, SEQ ID NO. 31 , SEQ ID NO. 35, or SEQ ID NO. 39.

[0151] The first antigen binding domain may comprise i) a heavy chain comprising a sequence comprising SEQ ID NO. 22 or a sequence comprising at least 70%, 80%, 85%, 90%, 95% or 99% sequence identity thereto, and ii) a light chain comprising a sequence comprising SEQ ID NO. 24 or a sequence comprising at least 70%, 80%, 85%, 90%, 95% or 99% sequence identity thereto.

[0152] The first antigen binding domain may comprise i) a heavy chain comprising a sequence selected from one of SEQ ID NO. 22, SEQ ID NO. 26, SEQ ID NO. 30, SEQ ID NO. 34, or SEQ ID NO. 38 and ii) a light chain variable (VL) region comprising a sequence selected from one of SEQ ID NO. 24, SEQ ID NO. 28, SEQ ID NO. 32, SEQ ID NO. 36, or SEQ ID NO. 40.

[0153] The second antigen binding domain may comprise i) a heavy chain variable (VH) region comprising a HC CDR1 comprising SEQ ID NO. 2, a HC CDR2 comprising SEQ ID NO. 3, a HC CDR3 comprising SEQ ID NO. 4, and ii) a light chain variable (VL) region comprising a LC CDR1 comprising SEQ ID NO. 7, a LC CDR2 comprising SEQ ID NO. 8, a LC CDR3 comprising SEQ ID NO. 9.

[0154] The second antigen binding domain may comprise i) a heavy chain variable (VH) region comprising SEQ ID NO. 1 and ii) a light chain variable (VL) region comprising SEQ ID NO. 6.

[0155] The second antigen binding domain may comprise a heavy chain variable (VH) region comprising SEQ ID NO. 1 or a sequence comprising 85%, 90%, 95% or 99% sequence identity thereto.

[0156] The second antigen binding domain may comprise a light chain variable (VL) region comprising SEQ ID NO. 6. or a sequence comprising 85%, 90%, 95% or 99% sequence identity thereto.

[0157] The percentage sequence identity recited herein for any of the sequences maybe selected from at least 70%, 75%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity thereto.

[0158] The bispecific binding molecule may comprise a first and second antigen binding domain wherein the first antigen binding domain comprises i) a heavy chain variable (VH) region comprising SEQ ID NO. 21 or a sequence comprising at least 90%, 92%, 95%, 97% or 99% sequence identity thereto, and ii) a light chain variable (VL) region comprising SEQ ID NO. 23 or a sequence comprising at least 80%, 85%, 90%, 92%, 95%, 97% or 99% sequence identity thereto, and the second antigen binding domain comprises i) a heavy chain variable (VH) region comprising a HC CDR1 comprising SEQ ID NO. 2, a HC CDR2 comprising SEQ ID NO. 3, a HC CDR3 comprising SEQ ID NO. 4, and ii) a light chain variable (VL) region comprising a LC CDR1 comprising SEQ ID NO. 7, a LC CDR2 comprising SEQ ID NO. 8, a LC CDR3 comprising SEQ ID NO. 9.

[0159] The bispecific binding molecule may comprise a first and second antigen binding domain wherein the first antigen binding domain comprises i) a heavy chain variable (VH) region comprising SEQ ID NO. 21 or a sequence comprising at least 90%, 92%, 95%, 97% or 99% sequence identity thereto, and ii) a light chain variable (VL) region comprising SEQ ID NO. 23 or a sequence comprising at least 80%, 85%, 90%, 92%, 95%, 97% or 99% sequence identity thereto, and the second antigen binding domain comprises i) a heavy chain variable (VH) region comprising SEQ ID NO. 1 and ii) a light chain variable (VL) region comprising SEQ ID NO. 6.

[0160] CD7 (cluster of differentiation 7) is a transmembrane glycoprotein and a member of the immunoglobulin supergene family. CD7 plays an important role in T cell and T cell / B-cell interactions during early lymphoid development. CD7 is found expressed on T cells and natural killer cells. Wild type human CD7 (UniProt Accession No. P09564). The amino acid sequence forwild type human CD7 is shown below (SEQ ID No. 41).

[0161] AQGRTFSVLLARLMVTAQVLPRGAAVSPLHDCPRGSLRQHHLLHQRGPAWDLPEAARATA PRHHLLRGRGGAHYGQTVPGPHRLLRVPGQPDYHHAPPAAVGHWHLHLPGHHGGQCLRLR HPGPGDRGTVPRMAQ

[0162] Unless otherwise specified, the term CD7 as used herein refers to human CD7. CD7 is also known as “GP40”, “TP41 ” or “T-Cell Leukemia Antigen”, and these terms are used interchangeably and include variants and isoforms of human CD7.

[0163] The terms "CD7 binding molecule / protein / polypeptide / agent / moiety”, "CD7 antigen binding molecule molecule / protein / polypeptide / agent / moiety”, “anti-CD7 antibody”, “anti-CD7 antibody fragment”, “anti-CD7 antibody or antigen binding portion thereof’ or“CD7 antibody” all refer to a molecule capable of specifically binding to the human CD7 antigen.

[0164] CD33 also known as sialic acid-binding Ig-like lectin 3 is an innate immune receptorthat is known to be expressed on myeloid cells. CD33 is a 67 kDa plasma membrane protein that binds to sialic acid and mediates inhibitory signalling via tyrosine phosphatases. CD33 is a member of the sialic acid-binding Ig-like lectin (SIGLEC) family. CD33 expression has also been reported on a number of malignant cells. It will be apparent to the skilled person that each protein may have a number of alternative names by which each protein is known. For example (non- exhaustive): CD33 may be known as: Siglec-3, sialic acid binding Ig-like lectin 3, SIGLEC3, SIGLEC-3, gp67, p67. CD56 may be known as: Neural cell adhesion molecule (NCAM), NCAM1 , MSK39, NCAM, neural cell adhesion molecule 1. Wild type human CD33 has been described (see e.g. UniProt Accession No. P20138). The amino acid sequence forwild type human CD33 is shown below (SEQ ID No. 42). DPNFWLQVQESVTVQEGLCVLVPCTFFHPIPYYDKNSPVHGYWFREGAIISRDSPVATNKLD QEVQEETQGRFRLLGDPSRNNCSLSIVDARRRDNGSYFFRMERGSTKYSYKSPQLSVHVTD LTHRPKILIPGTLEPGHSKNLTCSVSWACEQGTPPIFSWLSAAPTSLGPRTTHSSVLIITPRPQD HGTNLTCQVKFAGAGVTTERTIQLNVTYVPQNPTTGIFPGDGSGKQETRAGWHGAIGGAGV TALLALCLCLIFFIVKTHRRKAARTAVGRNDTHPTTGSASPKHQKKSKLHGPTETSSCSGAAPT VEMDEELHYASLNFHGMNPSKDTSTEYSEVRTQ

[0165] CD33 plays a role in the regulation of cellular calcium influx necessary for the development, differentiation, and activation of B-lymphocytes.

[0166] Unless otherwise specified, the term CD33 as used herein refers to human CD33. CD33 is also known as “Membrane Spanning 4-Domains A1 ”, “Bp35” or “FMC7”, and these terms are used interchangeably and include variants and isoforms of human CD33.

[0167] The terms "CD33 binding molecule / protein / polypeptide / agent / moiety”, "CD33 antigen binding molecule molecule / protein / polypeptide / agent / moiety”, “anti-CD33 antibody”, “anti-CD33 antibody fragment”, “anti-CD33 antibody or antigen binding portion thereof’ or “CD33 antibody” all refer to a molecule capable of specifically binding to the human CD33 antigen.

[0168] The present invention relates to a bispecific binding molecule which targets at least two antigens, wherein said antigens are CD7 and CD33. Bispecific refers to a binding molecule which binds to two different antigens, i.e. CD33 and CD7. The bispecific molecule is a heterodimeric molecule that binds both CD7 and CD33 antigen. The bispecific binding molecule may comprise an antibody or antigen binding fragment or portion thereof, wherein the antibody or antigen binding fragment or portion thereof targets at least two antigens, wherein said antigens are CD7 and CD33. The bispecific binding molecule may comprise a first antibody or antigen binding fragment or portion thereof, and a second antibody or antigen binding fragment or portion thereof wherein the first antibody or antigen binding fragment or portion thereof targets CD7, and the second antibody or antigen binding fragment or portion thereof targets CD33. The bispecific binding molecules may comprise further antigen binding domains for example the binding molecule may be multispecific e.g., trispecific. The further antigen binding domain may be provided as an antibody or antigen binding fragment or portion thereof.

[0169] The terms “antigen(s)” and “epitope(s)” are well established in the art and refer to the portion of a protein or polypeptide which is specifically recognized by a component of the immune system, e.g. an antibody or a T-cell I B-cell antigen receptor. As used herein, the term “antigen(s)” encompasses antigenic epitopes, e.g. fragments of antigens which are recognized by, and bind to, immune components. Epitopes can be recognized by antibodies in solution, e.g. free from other molecules. Epitopes can also be recognized by T-cell antigen receptors when the epitope is associated with a class I or class II major histocompatibility complex molecule.

[0170] The term “epitope” or “antigenic determinant” refers to a site on the surface of an antigen to which an immunoglobulin, antibody or antigen-binding fragment thereof specifically binds. Generally, an antigen has several or many different epitopes and reacts with many different antibodies. The term “specifically” includes linear epitopes and conformational epitopes.

[0171] Epitopes within protein antigens can be formed both from contiguous amino acids (usually a linear epitope) or non-contiguous amino acids juxtaposed by tertiary folding of the protein (usually a conformational epitope). Epitopes formed from contiguous amino acids are typically, but not always, retained on exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. An epitope typically includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14 or 15 amino acids in a unique spatial conformation. Methods for determining what epitopes are bound by a given antibody or antigen-binding fragment thereof (i.e., epitope mapping) are well known in the art and include, for example, immunoblotting and immunoprecipitation assays, wherein overlapping or contiguous peptides are tested for reactivity with a given antibody or antigen-binding fragment thereof. Competition assays can also be used to determine if a test antibody binds to the same epitope as a reference antibody. Suitable competition assays are mentioned elsewhere herein and also shown in the examples. In some aspects, the epitope to which an antibody or antigen-binding fragment thereof binds can be determined by, e.g, NMR spectroscopy, X-ray diffraction crystallography studies, ELISA assays, hydrogen / deuterium exchange coupled with mass spectrometry (e.g., liquid chromatography electrospray mass spectrometry), array-based oligo-peptide scanning assays, mutagenesis mapping (e.g, site-directed mutagenesis mapping), and / or in silico modelling.

[0172] A bispecific binding molecule of the invention, including a multispecific, e.g. trispecific, binding agent described herein, "which binds" or is “capable of binding” both CD7 and CD33, is one that binds both of the antigens CD7 and CD33 with sufficient affinity such that the bispecific antibody or antigen binding portion thereof is useful as a therapeutic agent in targeting a cell or tissue expressing the antigens CD7 and CD33 as described herein. The binding molecules may target CD7 and CD33 with different affinity, e.g. some binding molecules of the invention may target CD7 with a higher affinity than CD33, some binding molecules of the invention may target CD7 with a lower affinity than CD33. Binding molecules of the invention may target CD7 and CD33 with approximately the same affinity. The binding molecule may be capable of selectively targeting cells expressing CD7 and CD33, compared to cells expressing CD7 or cells expressing CD33. As such the binding molecule can be used to specifically target dual expressing CD7+ CD33+ cells. Dual expressing CD7+ CD33+ cells refers to cells which express both CD7 and CD33, the cells may also express further cell surface markers. In certain embodiments, the bispecific binding molecule specifically binds to CD7 and CD33 antigens that are cell surface expressed. As used herein, the expression “cell surface-expressed” means one or more CD7 and / or CD33 protein(s) that is / are expressed on the surface of a cell in vitro or in vivo, such that at least a portion of a CD7 and / or a CD33 protein is exposed to the extracellular side of the cell membrane and is accessible to the bispecific antibody of antigen binding fragments thereof of the invention.

[0173] “Specifically binds", "specific binding" or "selective binding" means that the binding is selective for the antigen and can be discriminated from unwanted or non-specific interactions. The ability of an antigen binding moiety to bind to a specific antigenic determinant can be measured either through an enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to one of skill in the art, e.g. surface plasmon resonance (SPR) technique (analyzed on a BIAcore instrument), and traditional binding assays. The binding reaction may be shown with reference to a negative control test using an antibody of unrelated specificity.

[0174] The term "antibody" as used herein refers to an immunoglobulin (Ig) protein that is capable of binding an antigen. In particular, the term "antibody" as used herein broadly refers to any polypeptide comprising complementarity determining regions (CDRs) that confer specific binding affinity of the polypeptide for an antigen. The term antibody as used herein encompasses polyclonal and monoclonal antibody preparations.

[0175] As antibodies can be modified in a number of ways, the term "antibody" should be construed as covering antibody fragments, derivatives, functional equivalents and homologues of antibodies, including any polypeptide comprising an immunoglobulin binding domain. The term “antibody” should also be construed as covering antibody mimetics, such as, but not limited to, cyclic peptides, for example bicyclic peptides, cysteine knots and anticalins etc. The antibody may be any immunoglobulin (Ig) molecule, or antigen binding portion / fragment thereof, comprised of four polypeptide chains, two heavy (H) chains and two light (L) chains, or any functional fragment, mutant, variant, or derivation thereof, which retains the essential epitope binding features of an Ig molecule. Such fragments are known in the art for example F(ab')2, Fab, Fab’, Fv, scFv, heavy chain, light chain, variable heavy (VH), variable light (VL) chain, CDR region, single VH or VL domain, maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, and bis-scFv, and polypeptides that contain at least a portion of an immunoglobulin that is sufficient to confer specific antigen binding to the polypeptide. Therefore, an antibody fragment comprises an antigen binding portion. The present invention extends to such antibody fragments. The term "monoclonal antibody" refers to an antibody obtained from a single close of cells or cell line. The individual antibodies are identical and / or bind the same epitope. Unlike polyclonal antibodies, which include different antibodies directed against different epitopes, each monoclonal antibody of in a preparation is directed against a single epitope.

[0176] The terms “polypeptide(s)” and “protein(s)” are used interchangeably throughout the application and denote at least two covalently attached amino acids, thus may signify proteins, polypeptides, oligopeptides, peptides, and fragments thereof. The protein may be made up of naturally occurring amino acids and peptide bonds, or synthetic peptidomimetic structures. Hence, “amino acid(s)” or “peptide residue(s)”, as used herein, denote both naturally occurring and synthetic amino acids. In some cases, the immunoglobulin proteins of the present invention may be synthesized using any in vivo or in vitro protein synthesis technique known in the art.

[0177] The bispecific binding molecules of the present invention may comprise a bispecific antibody, or a bispecific antigen binding fragment thereof. The bispecific antibody or antigen binding fragment may comprise two antigen binding domains wherein the antigen binding domains comprise the same format or wherein the antigen binding domains comprise different formats. The bispecific binding molecule may comprise a bispecific antibody. The bispecific binding molecule may comprise a bispecific antigen binding fragment. An antigen binding fragment may only comprise one antigen binding domain, as such a bispecific antigen binding fragment may comprise two antigen binding fragments linked in any suitable manner.

[0178] A full-length antibody comprises two heavy (H) chains and two light (L) chains. Each heavy chain is comprised of a heavy chain variable region or domain and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CH1 , CH2 and CH3. Each light chain is comprised of a light chain variable region or domain and a light chain constant region. The light chain constant region is comprised of one domain, CL.

[0179] The heavy chain and light chain variable regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each heavy chain and light chain variable region is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1 , CDR1 , FR2, CDR2, FR3, CDR3, FR4.

[0180] Immunoglobulin molecules can be subdivided into various types class and subclass. In humans the classes (isotype) of immunoglobulin include IgG, IgM, IgA, IgE, and IgD. The immunoglobulin classes are distinguished by the type of heavy chain they contain. IgG molecules possess heavy chains known as y-chains; IgM have p-chains; IgA have a-chains; IgE have s-chains; and IgD have 6-chains. The antibody or antigen binding fragment may be IgG type. There are two types of light chain that can be present these are kappa(K) and lambda (A). The antibodies or antigen binding fragments of the invention may comprise either K light chain or A light chain. There are four types of heavy chain the a (alpha) (IgA), y (gamma) (lgG1 , lgG2, lgG3, lgG4), 6 (delta) (IgD), s (epsilon) (IgE) and p (mu) (IgM) heavy chains, or their equivalents in other species (for example rodent, canine etc). Full-length immunoglobulin “light chains” (usually of about 25 kDa or 214 amino acids long) consist of a variable region of approximately 110 amino acids at the NH2-terminus and a kappa or lambda constant region at the COOH- terminus. Full-length immunoglobulin “heavy chains” (usually of about 50 kDa or 446 amino acids long), likewise consist of a variable region (of about 116 amino acids) and one of the aforementioned heavy chain constant regions, e.g., gamma (of about 330 amino acids).

[0181] Light or heavy chain variable regions are generally composed of a “framework” region (FR) interrupted by three hypervariable regions, also called CDRs. The extent of the framework region and CDRs have been precisely defined. The sequences of the framework regions of different light and heavy chains are relatively conserved within a species. The framework region of an antibody, i.e., the combined framework regions of the constituent light and heavy chains, serves to position and align the CDRs. The CDRs are primarily responsible for binding to an epitope of an antigen. There are three CDRs in each of the variable regions of the heavy chain and the light chain, which are designated CDR1 , CDR2 and CDR3, for each of the variable regions. The term "CDR set" refers to a group of three CDRs that occur in a single variable region capable of binding the antigen. The exact boundaries of these CDRs can be defined differently according to different systems known in the art.

[0182] Heavy chain CDRs are designated HCDR1 , HCDR2 and HCDR3. Light chain CDRs are designated LCDR1 , LCDR2 and LCDR3.

[0183] The antibody may be comprised of four polypeptide chains, two heavy (H) chains and two light (L) chains, or any functional fragment, mutant, variant, or derivation thereof, which retains the essential epitope binding features of an Ig molecule. Such mutant, variant, or derivative antibody formats are known in the art.

[0184] The term "CDR" (complementarity-determining region) refers to the immunoglobulin hypervariable domains within an antibody or antigen binding fragment sequences. It is generally accepted that the CDRs determine the specific antibody binding. There are three CDRs in each of the variable regions of the heavy chain and the light chain, which are designated CDR1 , CDR2 and CDR3, for each of the variable regions. The term "CDR set" refers to a group of three CDRs that occur in a single variable region capable of binding the antigen. The exact boundaries of these CDRs can be defined differently according to different systems known in the art. The Kabat Complementarity Determining Regions (CDRs) are based on sequence variability and was originally derived from observations from sequence alignments of light chain A, K and heavy chain sequences for a number of antibodies and for the a, p, y, 6 chain sequences for a number of TCRs. Kabat numbering is the most commonly used (Kabat et al., (1971) Ann. NY Acad. Sci. 190:382-391 and Kabat, et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91- 3242). Chothia refers instead to the location of the structural loops (Chothia and Lesk J. Mol. Biol. 196:901 -917 (1987)). Another system is the ImMunoGeneTics (IMGT) numbering scheme. The IMGT numbering scheme is described in Lefranc et al., Dev. Comp. Immunol., 29, 185-203 (2005). These terms, which are recognized in the art, refer to a system of numbering amino acid residues which are more variable (i.e., hypervariable) than other amino acid residues in the heavy and light chain variable regions of an antibody, or an antigen binding portion.

[0185] Unless otherwise specified, the IMGT numbering system is used herein when referring to CDRs. The CDR sequences of the antigen binding region are herein provided according to the IMGT numbering scheme unless otherwise specified. The CDR sequences of the antigen binding regions according to the invention may also comprise the CDR sequences as defined by any antibody numbering scheme known in the art including Kabat, IMGT, Chothia, Martin, AHo. The ImMunoGeneTics (IMGT) unique numbering scheme (IMGT numbering scheme) is described in Lefranc et al., Dev. Comp. Immunol., 29, 185-203 (2005). The IMGT numbering scheme is based on alignment of germ-line V genes and covers the entire variable region positions 1 to 128 based on the V-gene sequence alignment, with an insertion point between positions 111 and 112 in the CDR3 for lengths exceeding 13 amino acids. The Kabat Complementarity Determining Regions (CDRs) are based on sequence variability and was originally derived from observations from sequence alignments of light chain A, K and heavy chain sequences for a number of antibodies and for the a, p, y, 6 chain sequences for a number of TCRs (Kabat et al., (1971) Ann. NY Acad. Sci. 190:382-391 and Kabat, et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242). The Chothia scheme is a structure-based scheme and refers to the location of the structural loops (Chothia and Lesk J. Mol. Biol. 196:901 -917 (1987)) Martin a further structural numbering scheme similar to Chothia which comprises greater sequence variability in the reference dataset, also unconventional CDR lengths and deletions are taken into account in the numbering scheme. The AHo scheme comprises a large number of positions is supposed to account for all possible positions without the need for specifying insertion positions. These terms, which are recognized in the art, refer to a system of numbering amino acid residues which are more variable (i.e., hypervariable) than other amino acid residues in the heavy and light chain variable regions of an antibody, or an antigen binding portion. The skilled person would be able to determine the CDR sequences, as defined by any antibody numbering scheme known in the art including Kabat, IMGT, Chothia, Martin, AHo, from a full-length VH or VL sequence using standard techniques known in the art

[0186] The term "antibody" is not only inclusive of antibodies generated by methods comprising immunisation, but also includes any polypeptide, e.g., a recombinantly expressed polypeptide, which is made to encompass at least one CDR capable of specifically binding to an epitope on an antigen of interest. Hence, the term applies to such molecules regardless whether they are produced in vitro, in cell culture, or in vivo. Methods of producing polyclonal and monoclonal antibodies are known in the art.

[0187] It is possible to take monoclonal and other antibodies and use techniques of recombinant DNA technology to produce other antibodies or chimeric molecules which generally retain the specificity of the original antibody. Such techniques may involve introducing the CDRs into a different immunoglobulin framework, or grafting variable regions onto a different immunoglobulin constant regions. Alternatively, a hybridoma or other cell producing an antibody molecule may be subject to genetic mutation or other changes, which may or may not alter the binding specificity of antibodies produced.

[0188] The antibody or antigen-binding fragment thereof may be chimeric, human or humanized. A “chimeric antibody” is a recombinant protein that contains the variable domains including the CDRs of an antibody derived from one species, for example a murine antibody, while the constant domains of the antibody molecule are derived from those of a different species, for example a human antibody. Methods to humanise antibodies include CDR grafting based on framework regions homology and antibody resurfacing. Human or humanised antibodies or antigen-binding fragments are most desirable for use in antibody therapies, as such molecules would elicit little or no immune response in the human subject. The antibody or antigen-binding fragment thereof is a humanized antibody or antigen-binding fragment thereof. The first and / or second binding domains of the bispecific molecule may be humanized. The CD7 binding arm of the bispecific binding molecule may be a humanized molecule.

[0189] A “humanized antibody” or fragment thereof is a recombinant protein in which the CDRs from an antibody from one species; e.g., a rodent, canine, feline antibody, are transferred from the heavy and light variable chains of the rodent, canine or feline antibody into human heavy and light variable domains (e.g., framework region sequences). The constant domains of the antibody molecule are derived from those of a human antibody. In certain embodiments, a limited number of framework region amino acid residues from the parent (e.g., rodent) antibody may be substituted into the human antibody framework region sequences. In an embodiment the CDRs disclosed herein may be transferred from the heavy and light variable chains of the antibody disclosed herein into the heavy and light variable domains (e.g., framework region sequences) of a different species.

[0190] The antibody or antigen-binding fragment may comprise a monoclonal antibody or antigenbinding fragment thereof.

[0191] The binding molecules of the present invention may include antibodies or antigen binding fragments thereof. The binding molecules of the invention comprise a first and second antigen binding domain, the first antigen binding domain may comprise an antibody or an antigen binding fragment thereof, the second antigen binding domain may comprise an antibody or an antigen binding fragment thereof. The antigen-binding fragments may be selected from any fragment capable of binding the antigen or antigenic fragment of interest. Exemplary antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, F(ab')3, Fabc, Fd, single chain Fv (scFv), (scFv)2, Fv, scFv-Fc, heavy chain only antibody, diabody, tetrabody, triabody, minibody, antibody mimetic protein, single domain antibody, e.g. a VH. Thus, the antigen-binding fragment may comprise or consist of any of these fragments. The term antigen binding domain as used herein refers to a polypeptide capable of binding an antigen.

[0192] Antigen-binding fragments derived from an antibody, including single-chain antibodies, may comprise the variable region(s) alone or in combination with the entire, or parts of the, following: a heavy chain constant domain, or a portion thereof, e.g. a CH1 , CH2, CH3, transmembrane, and / or cytoplasmic domain, on the heavy chain, and a light chain constant domain, e.g. a Ckappa or Clambda domain, or portion thereof on the light chain. Also included in the present disclosure are any combinations of variable region(s) and CH1 , CH2, CH3, Ckappa, Clambda, transmembrane and cytoplasmic domains.

[0193] The antibody or antigen-binding fragment may comprise a CH2 domain. The CH2 domain is for example located at the N- terminus of the CH3 domain, as in the case in a human IgG molecule. The CH2 domain of the antibody may be the CH2 domain of human lgG1 , lgG2, lgG3, or lgG4, e.g., the CH2 domain of human lgG1 . The sequences of human IgG domains are known in the art.

[0194] The antibody or antigen-binding fragment may comprise an immunoglobulin hinge region, or part thereof, at the N-terminus of the CH2 domain. The immunoglobulin hinge region allows the two CH2-CH3 domain sequences to associate and form a dimer. The hinge region, or part thereof, may be a human IgG 1 , lgG2, lgG3 or lgG4 hinge region, or part thereof. For example, the hinge region, or part thereof, may be an IgG 1 hinge region, or part thereof. The sequence of the CH3 domain is not particularly limited. The CH3 domain may be a human immunoglobulin G domain, such as a human lgG1 , lgG2, lgG3, or lgG4 CH3 domain, e.g. a human lgG1 CH3 domain.

[0195] The antibody or antigen-binding fragment may comprise a human lgG1 , lgG2, lgG3, or lgG4 constant region. The sequences of human IgG 1 , lgG2, lgG3, or lgG4 CH3 domains are known in the art. The antibody or antigen-binding fragment may comprise a non-human IgG constant region, e.g., a rabbit lgG1 constant region.

[0196] The antigen binding fragment according to the present invention include functional fragments of an antibody. A functional fragment of an antibody is a fragment which still retains the ability to bind to a target antigen, as such the functional fragment comprises an antigen binding domain. Examples of functional fragments of an antibody include Fab, Fab’, F(ab)2, scFv, (scFv), heavy chain only antibody, single domain antibody, nanobody, these terms are known in the art and describe specific formats of antigen binding fragments.

[0197] Antigen-binding fragments derived from an antibody, including single-chain antibodies, may comprise the variable region(s) alone or in combination with the entire, or parts of the, following: a heavy chain constant domain, or a portion thereof, e.g. a CH1 , CH2, CH3, transmembrane, and / or cytoplasmic domain, on the heavy chain, and a light chain constant domain, e.g. a Ckappa or Clambda domain, or portion thereof on the light chain. Also included in the present disclosure are any combinations of variable region(s) and CH1 , CH2, CH3, Ckappa, Clambda, transmembrane and cytoplasmic domains.

[0198] Fv fragments (~25kDa) consist of the two variable domains, VH and VL. Naturally, VH and VL domain are non-covalently associated via hydrophobic interaction and tend to dissociate. However, stable fragments can be engineered by linking the domains with a hydrophilic flexible linker to create a single chain Fv (scFv).

[0199] The smallest antigen-binding fragment is the single variable fragment, namely the variable heavy (VH) or variable light (VL) chain domain. VH and VL domains respectively are capable of binding to an antigen. They are generally referred to as a “single domain antibody” or “immunoglobulin single variable domain”. A single domain antibody (~12 to 15 kDa) has thus either the VH or VL domain. Antigen-binding single VH domains have also been identified from, for example, a library of murine VH genes amplified from genomic DNA from the spleens of immunized mice and expressed in E. coli (Ward et al., 1989, Nature 341 : 544-546). Ward et al. named the isolated single VH domains "dAbs," for "domain antibodies." The term "dAb" or “sdAb” (for single domain antibody) generally refers to a single immunoglobulin variable domain (VH, VHH or VL) polypeptide that specifically binds antigen. For use in therapy, human single domain antibodies are preferred over camelid derived VHH, primarily because they are not as likely to provoke an immune response when administered to a patient.

[0200] A "Fab molecule" (fragment antigen binding) as such a Fab domain refers to a protein consisting of the VH and CHI domain of the heavy chain (the "Fab heavy chain") and the VL and CL domain of the light chain (the "Fab light chain") of an immunoglobulin. In certain embodiments the Fab light chain and Fab heavy chain in the Fab construct are linked by a polypeptide sequence to yield a single chain Fab (scFab).

[0201] A “Fab” or “Fab fragment” comprises an antigen-binding domain comprising or consisting of one constant and one variable domain of each of the heavy and the light chains. For example a Fab contains the constant domain (CL) of the light chain and the first constant domain (CHI) of the heavy chain along with the variable domains VL and VH on the light and heavy chains respectively. The variable domains comprise the complementarity determining loops (CDR, also referred to as hypervariable region) that are involved in antigen binding. A Fab’ comprises an antigen-binding domain comprising or consisting of one constant and one variable domain of each of the heavy and the light chains and the thiol group which forms the disulphide bridge between two heavy chains in a full-length antibody. As such Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CHI domain including one or more cysteines from the antibody hinge region.

[0202] As used herein, the term "single-chain" refers to a molecule comprising amino acid monomers linearly linked by peptide bonds. In certain embodiments, one of the antigen binding moieties, e.g., antigen binding polypeptide construct, is a single-chain Fab molecule, i.e. a Fab molecule wherein the Fab light chain and the Fab heavy chain are connected by a peptide linker to form a single peptide chain. In a particular such embodiment, the C-terminus of the Fab light chain is connected to the N-terminus of the Fab heavy chain in the single- chain Fab molecule. Fv fragments (~25kDa) consist of the two variable domains, VH and VL. Naturally, VH and VL domain are non-covalently associated via hydrophobic interaction and tend to dissociate. However, stable fragments can be engineered by linking the domains with a hydrophilic flexible linkerto create a single chain Fv (scFv). In certain other embodiments, one of the antigen binding moieties is a single-chain Fv molecule (scFv).

[0203] Single chain fragment variable (scFv) antibody fragments comprise the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. In one embodiment, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding. In one embodiment the scFv is a fusion protein comprising the heavy chain variable region and the light chain variable region linked via a peptide linker. The antigen binding fragment may comprise a Fab, Fab’, F(ab)2, scFv, (scFv), heavy chain only antibody, single domain antibody, or nanobody. In an embodiment the antigen binding fragment comprises a Fab. In an embodiment the antigen binding fragment comprises a Fab’. In an embodiment the antigen binding fragment comprises an scFv. In an embodiment the bispecific antibody or antigen binding fragment, comprises a first and second antigen binding domain, wherein said first and / or second antigen binding domain comprises a Fab, Fab’, F(ab)2, scFv, (scFv), heavy chain only antibody, single domain antibody, or nanobody. In an embodiment the first antigen binding domain comprises a Fab, Fab’, F(ab)2, scFv, (scFv), heavy chain only antibody, single domain antibody, or nanobody. In an embodiment the second antigen binding domain comprises a Fab, Fab’, F(ab)2, scFv, (scFv), heavy chain only antibody, single domain antibody, or nanobody. In an embodiment the first and second antigen binding domain comprises a Fab or a Fab’. In an embodiment the first and second antigen binding domain comprises a Fab. In an embodiment the first and second antigen binding domain comprises or consists of a Fab. In an embodiment the first and second antigen binding domain comprises or consists of a Fab. In an embodiment the first antigen binding domain comprises or consists of a Fab. In an embodiment the second antigen binding domain comprises or consists of a Fab. In an embodiment the first and second antigen binding domain each comprise or consist of a Fab. In an embodiment the first and second antigen binding domain comprises an scFv.

[0204] In an embodiment the bispecific binding molecule may comprise a first and second antigen binding domain wherein the first antigen binding domain comprises i) a heavy chain variable (VH) region comprising SEQ ID NO. 21 or a sequence comprising at least 90%, 92%, 95%, 97% or 99% sequence identity thereto, and ii) a light chain variable (VL) region comprising SEQ ID NO. 23 or a sequence comprising at least 80%, 85%, 90%, 92%, 95%, 97% or 99% sequence identity thereto, and the second antigen binding domain comprises i) a heavy chain variable (VH) region comprising SEQ ID NO. 1 and ii) a light chain variable (VL) region comprising SEQ ID NO. 6 wherein the first antigen binding domain comprises or consists of a Fab and the second antigen binding domain comprises or consists of a Fab.

[0205] The bispecific binding molecule, antibody or antigen binding fragment thereof may comprise an Fc domain. The term "Fc" or "Fc domain" or "Fc region" or "Fc construct" herein is used to define a C-terminal region of an immunoglobulin heavy chain. The term includes native sequence Fc regions and variant Fc regions.

[0206] "Fc region", as used herein, generally refers to a dimer complex comprising the C-terminal polypeptide sequences of an immunoglobulin heavy chain, wherein a C-terminal polypeptide sequence is that which is obtainable by papain digestion of an intact antibody. The Fc region may comprise native or variant Fc sequences.

[0207] The Fc sequence of an immunoglobulin generally comprises two constant domains, a CH2 domain and a CH3 domain, and optionally comprises a CH4 domain. By "Fc polypeptide" herein is meant one of the polypeptides that make up an Fc region. An Fc polypeptide may be obtained from any suitable immunoglobulin, such as IgG 1 , lgG2, lgG3, or lgG4 subtypes, IgA, IgE, IgD or IgM. In some embodiments, an Fc polypeptide comprises part or all of a wild type hinge sequence (generally at its N terminus). In some embodiments, an Fc polypeptide does not comprise a functional or wild type hinge sequence. The antibody may comprise a CH2 domain. The CH2 domain is for example located at the N- terminus of the CH3 domain, as in the case in a human IgG molecule. The CH2 domain of the antibody is in one embodiment the CH2 domain of human lgG1 , lgG2, lgG3, or lgG4, e.g the CH2 domain of human lgG1. The sequences of human IgG domains are known in the art.

[0208] Thus, bispecific binding molecule, antibody or antigen binding fragment thereof may comprise an immunoglobulin hinge region, or part thereof, at the N-terminus of the CH2 domain. The immunoglobulin hinge region allows the two CH2-CH3 domain sequences to associate and form a dimer. In one embodiment, the hinge region, or part thereof, is a human IgG 1 , lgG2, lgG3 or lgG4 hinge region, or part thereof. For example, the hinge region, or part thereof, is an lgG1 hinge region, or part thereof.

[0209] The first and / or second antibody or antigen-binding fragment may comprise an Fc domain. For example, one of the first or second antibody or antigen-binding fragment may comprise an Fc domain. For example, the first and / or second antibody or antigen-binding fragment may comprise i) a Fab, F(ab')2, Fv, a single chain Fv fragment (scFv) and / or a single domain antibody and ii) an Fc domain. For example, one of the first or second antibody or antigen-binding fragment may comprise i) a Fab, F(ab')2, Fv, a single chain Fv fragment (scFv) and / or a single domain antibody and ii) an Fc domain.

[0210] The bispecific binding molecule, antibody or antigen binding fragment thereof may comprise an Fc region. The bispecific molecule may comprise at least one Fc region. Where the bispecific molecule comprises a first and a second antibody or antigen binding fragment, the first and / or the second antibody or antigen binding fragment may comprise an Fc region as described herein. The Fc region may be any suitable Fc region for example a human Fc region.

[0211] Thus, bispecific binding molecule, antibody or antigen binding fragment thereof may comprise a CH3 domain. The sequence of the CH3 domain is not particularly limited. In one embodiment, the CH3 domain is a human immunoglobulin G domain, such as a human lgG1 , lgG2, lgG3, or lgG4 CH3 domain, e.g. a human lgG1 CH3 domain.

[0212] Thus, the bispecific binding molecule, antibody or antigen binding fragment thereof may comprise a human lgG1 , lgG2, lgG3, or lgG4 constant region. The sequences of human lgG1 , lgG2, lgG3, or lgG4 CH3 domains are known in the art. An binding molecule, antibody or antigen binding fragment of the invention may comprise a non-human IgG constant region, e.g., a rabbit lgG1 constant region.

[0213] The Fc includes two Fc polypeptides each having a CH3 domain for dimerization. The N- terminal end of each Fc polypeptide is linked to the C-terminus of one of the antigen binding polypeptide constructs with or without a linker.

[0214] An Fc region can mediate downstream effector functions via interaction with Fc-receptors found on immune cells or with C1q, the recognition molecule of the complement system. The antibody or antigen-binding fragment may comprise an Fc region may be capable of interacting with an Fc receptor. The bispecific molecule may comprise an Fc region may be capable of interacting with an Fc receptor and eliciting a downstream effector function. Effector functions refer to downstream immune effector mechanisms such as but not limited to antibody-dependent cytotoxicity (ADCC), antibody dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), antibody-dependent intracellular neutralization and / or immunomodulatory functions.

[0215] Fc receptors (FcRs) are key immune regulatory receptors connecting the antibody mediated (humoral) immune response to cellular effector functions. Receptors for all classes of immunoglobulins have been identified, including FcyR (IgG), FcsRI (IgE), FcaRI (IgA), FcpR (IgM) and FcbR (IgD). There are three classes of receptors for human IgG found on leukocytes: CD64 (FcyRI), CD32 (FcyRlla, FcyRllb and FcyRllc) and CD16 (FcyRllla and FcyRlllb). FcyRI is classed as a high affinity receptor (nanomolar range KD) while FcyRI I and FcyRI 11 are low to intermediate affinity (micromolar range KD).

[0216] "Antibody-dependent cell-mediated cytotoxicity" and "ADCC" refer to a cell- mediated reaction in which nonspecific cytotoxic cells that express Fc receptors (FcRs) (e.g. Natural Killer (NK) cells, neutrophils, and macrophages) recognize bound antibody on a target cell and subsequently cause lysis of the target cell.

[0217] "Complement dependent cytotoxicity" and "CDC" refer to the lysing of a target in the presence of complement. The complement activation pathway is initiated by the binding of the first component of the complement system (Clq) to a molecule (e.g. an antibody) complexed with a cognate antigen.

[0218] "Antibody-dependent cellular phagocytosis and "ADCP" refer to the destruction of target cells via monocyte or macrophage-mediated phagocytosis.

[0219] The bispecific binding molecule may comprise a human IgG Fc with orwithout effector function.

[0220] In antibody ADCC, FcyRs on the surface of effector cells (natural killer cells, macrophages, monocytes and eosinophils) bind to the Fc region of an IgG which itself is bound to a target cell. Upon binding a signalling pathway is triggered which results in the secretion of various substances, such as lytic enzymes, perforin, granzymes and tumour necrosis factor, which mediate in the destruction of the target cell. The level of ADCC effector function various for IgG subtypes. Although this is dependent on the allotype and specific FcyRs in simple terms ADCC effector function is high for human lgG1 and lgG3, and low for lgG2 and lgG4.

[0221] FcyRs bind to IgG asymmetrically across the hinge and upper CH2 region. Knowledge of the binding site has resulted in engineering efforts to modulate IgG effector functions.

[0222] The bispecific binding molecule, antibody or antigen binding fragment thereof of the invention may have an Fc region with effector function, with enhanced effector function or with reduced effector function. In particular, the binding molecule, antibody or antigen binding fragment thereof may include a modified Fc region.

[0223] The bispecific molecule may comprise a modified Fc. The modified Fc may be modified in terms of amino acid sequence compared to a wild-type Fc region.

[0224] The Fc region may be modified to modulate the effect of the Fc region. For example the Fc region may be modified to enhance effector function. The bispecific molecule may comprise an Fc with enhanced effector function. The Fc region may be modified to reduce or abolish effector function.

[0225] The Fc region may be modified to improve certain properties, e.g. to reduce complement mediated effector function, to reduce FcyR- mediated effector functions, to improve half-life. The Fc region may be modified to enhance or reduce interaction of the Fc with one or more of the Fc receptors e.g. FcRn, C1q, TRIM21 , FcyRI, FcyRlla / b, FcyRllla).

[0226] The potency of antibodies can be increased by enhancement of the ability to mediate cellular cytotoxicity functions, such ADCC and ADCP. A number of mutations within the Fc domain have been identified that either directly or indirectly enhance binding of Fc receptors and significantly enhance cellular cytotoxicity: the mutations S239D / A330L / I332E (“3M”), F243L or G236A. Alternatively, enhancement of effector function can be achieved by modifying the glycosylation of the Fc domain, FcyRs interact with the carbohydrates on the CH2 domain and the glycan composition has a substantial effect on effector function activity. Afucosylated (non-fucosylated) antibodies, exhibit greatly enhanced ADCC activity through increased binding to FcyRllla.

[0227] Activation of ADCC and CDC may be desirable for some therapeutic antibodies, however, in some embodiments, an antibody that does not activate effector functions is preferred. Due to their lack of effector functions, lgG4 antibodies are the preferred IgG subclass for receptor blocking without cell depletion. However, lgG4 molecules can exchange half molecules in a dynamic process termed Fab-arm exchange. This phenomenon can occur between therapeutic antibodies and endogenous lgG4. The S228P mutation has been shown to prevent this recombination process allowing the design of lgG4 antibodies with a reduced propensity for Fabarm exchange. Fc engineering approaches have been used to determine the key interaction sites for the lgG1 Fc domain with Fey receptors and C1 q and then mutate these positions to reduce or abolish binding. Through alanine scanning the binding site of C1q to a region covering the hinge and upper CH2 of the Fc domain was identified. The CH2 domain of an antibody or fragment of the invention may comprise one or more mutations to decrease or abrogate binding of the CH2 domain to one or more Fey receptors, such as FcyRI, FcyRlla, FcyRllb, FcyRIII and / or to complement. CH2 domains of human IgG domains normally bind to Fey receptors and complement, decreased binding to Fey receptors is expected to decrease ADCC and decreased binding to complement is expected to decrease CDC activity of the antibody molecule.

[0228] Mutations to decrease or abrogate binding of the CH2 domain to one or more Fey receptors and / or complement are known in the art. An antibody molecule of the invention may comprise an Fc with modifications K322A / L234A / L235A or L234F / L235E / P331 S (“TM”), which almost completely abolish FcyR and C1q binding. An antibody molecule of the invention may comprise a CH2 domain, wherein the CH2 domain comprises alanine residues at EU positions 234 and 235 (positions 1 .3 and 1 .2 by IMGT numbering) ("LALA mutation"). Furthermore, complement activation and ADCC can be decreased by mutation of Pro329 (position according to EU numbering), e.g., to either P329A or P329G. The antibody molecule of the invention may comprise a CH2 domain, wherein the CH2 domain comprises alanine residues at EU positions 234 and 235 (positions 1.3 and 1.2 by IMGT numbering) and an alanine (LALA- PA) or glycine (LALA-PG) at EU position 329 (position 114 by IMGT numbering). Additionally or alternatively, an antibody molecule of the invention may comprise an alanine, glutamine or glycine at EU position 297. Modification of glycosylation on asparagine 297 of the Fc domain, which is known to be required for optimal FcR interaction may confer a loss of binding to FcRs; a loss of binding to FcRs has been observed in N297 point mutations. An antibody molecule of the invention may comprise an Fc with an N297A, N297G or N297Q mutation. An antibody molecule of the invention with an aglycosyl Fc domain may be obtained by enzymatic deglycosylation, by recombinant expression in the presence of a glycosylation inhibitor or following the expression of Fc domains in bacteria.

[0229] The Fc region may be modified to improve the half-life of the bispecific molecule. The Fc region may be modified to enhance interaction with FcRn receptor and thereby improve half-life of the bispecific molecule. IgG naturally persists for a prolonged period in the serum due to FcRn- mediated recycling, giving it a typical half-life of approximately 21 days. Half-life can be extended by engineering the pH-dependant interaction of the Fc domain with FcRn to increase affinity at pH 6.0 while retaining minimal binding at pH 7.4. The T250Q / M428L variant, conferred an approximately 2-fold increase in IgG half-life (assessed in rhesus monkeys), while the M252Y / S254T / T256E variant, gave an approximately 4-fold increase in IgG half-life (assessed in cynomolgus monkeys). Extending half-life may allow the possibility of decreasing administration frequency, while maintaining or improving efficacy. The Fc region may be modified to reduce interaction with FcyR receptor and thereby improve half-life of the bispecific molecule. An example of a modification that may be made to improve half-life is introduction of M252Y / S254T / T256E mutations in lgG1 CH2 domain, this modification is commonly referred to as a “YTE” variant.

[0230] The bispecific binding molecule, antibody or antigen binding fragment thereof may comprise an Fc which does not elicit downstream effector function, i.e., where its effector function has been knocked out. In such embodiments, the half-life of the compound may be increased. Methods to modify or modulate the Fc effector function are known in the art and may be achieved via site directed mutagenesis of the Fc region. The modulated Fc refers to a modulated activity compared to that of the wild-type Fc.

[0231] In one embodiment, the Fc is an lgG1 Fc construct, and lgG2 Fc construct, an lgG3 Fc construct, or an lgG4 Fc construct. In some embodiments, at least one CH3 domain has at least one amino acid modification that promotes the formation of a heterodimeric Fc with stability comparable to a wild-type homodimeric Fc. Exemplary modifications are described below.

[0232] Production of bispecific antibodies of the IgG type by co-expression of the two light and two heavy chains in a single host cell can be highly challenging because of the low yield of desired bispecific IgGs and the difficulty in removing closely related mispaired IgG contaminants. This reflects that heavy chains form homodimers as well as the desired heterodimers - the so-called heavy chain-pairing problem. Additionally, light chains can mispair with non-cognate heavy chains - the so-called light chain pairing problem. Consequently, coexpression of two antibodies can give rise to up to nine unwanted IgG species in addition to the desired bispecific antibody. Various approaches are described in the art in order to promote heterodimerisation, i.e. the formation of a certain bispecific antibody of interest for human therapy, thereby reducing the content of undesired homodimers in the resulting mixture. These approaches have been studied in relation to human or humanised antibodies designed to target disease in humans.

[0233] The homodimerisation of the two heavy chains in an IgG is mediated by the non-covalent interaction between the CH3 domains alone. Thus, CH3-CH3 interaction is the primary driver for Fc dimerisation.

[0234] It is furthermore well-known that when two CH3 domains interact with each other they meet in a protein-protein interface which comprises “contact” residues (also called contact amino acids, interface residues or interface amino acids). Contact amino acids of a first CH3 domain interact with one or more contact amino acids of a second CH3 domain. Contact amino acids are typically within 5.5 A (preferably within 4.5 A) of each other in the three-dimensional structure of an antibody. The interaction between contact residues from one CH3 domain and contact residues from a different CH3 domain may for instance be via Van der Waals forces, hydrogen bonds, water-mediated hydrogen bonds, salt bridges or other electrostatic forces, attractive interactions between aromatic side chains, disulfide bonds, or other forces known to one skilled in the art. The primary drive is thus hydrophobic interaction in the core and electrostatic interactions.

[0235] Approaches to interfere with the dimerisation of antibody heavy chains have been employed in the art to bias production of heterodimeric antibodies. Specific engineering in the CH3 domains was applied in order to favour heterodimerisation over homodimerisation. Examples of such engineering of the CH3-CH3 interface include the introduction of complementary protuberance and cavity mutations, also known as ‘knob-into-hole’ approaches as described for instance in WO9627011 incorporated by reference and J.B. Ridgway et al 'Knobs-into-holes' engineering of antibody CH3 domains for heavy chain heterodimerisation Protein Eng., 9 (1996), pp. 617-621 .

[0236] Generally, the method involves introducing a protuberance at the interface of a first polypeptide and a corresponding cavity in the interface of a second polypeptide, such that the protuberance can be positioned in the cavity so as to promote heteromultimer formation and hinder homomultimer formation. “Protuberances” or “knobs” are constructed by replacing small amino acid side chains from the interface of the first polypeptide with larger side chains (e.g. tyrosine or tryptophan). Compensatory “cavities” or “holes” of identical or similar size to the protuberances are created in the interface of the second polypeptide by replacing large amino acid side chains with smaller ones (e.g. alanine or threonine). The protuberance and cavity can be made by synthetic means such as altering the nucleic acid encoding the polypeptides or by peptide synthesis. Starting from a “knob” mutation (T366W) (Ridgway et al., supra) that disfavors CH3 homodimerisation, compensating “hole” mutations (T366S, L368A, and Y407V) (Atwell et al Stable heterodimers from remodeling the domain interface of a homodimer using a phage display library J. Mol. Biol., 270, pp. 26-35, 1997) were identified by phage display providing efficient pairing with the “knob” while disfavoring homodimerisation.

[0237] Several other successful strategies for heavy chain heterodimerisation, including electrostatic steering mutations (W02006 / 106905 and Gunasekaran et al Enhancing antibody Fc heterodimer formation through electrostatic steering effects: applications to bispecific molecules and monovalent IgG J. Biol. Chem., 285, pp. 19637-19646, 2010). This approach is based on electrostatic engineering of contact residues within the CH3-CH3 interface that are naturally charged. Mutations are introduced in the CH3 domains of heavy chains wherein naturally occurring charged amino acid contact residues are replaced by amino acid residues of opposite charge (i.e. a charge reversal strategy). This creates an altered charge polarity across the Fc dimer interface such that co-expression of electrostatically matched Fc chains supports favorable attractive interactions thereby promoting desired Fc heterodimer formation, whereas unfavorable repulsive charge interactions suppress unwanted Fc homodimer formation.

[0238] It has been described that within the human CH3-CH3 interface four unique charges residue pairs are involved in the domain-domain interaction. These are D356 / K439', E357 / K370', K392 / D399' and D399 / K409' (numbering according to Kabat, 1991) where residues in the first chain are separated from residues in the second chain by 7’ and where the prime Q indicates the residue numbering in the second chain). As the CH3-CH3 interface displays a 2-fold symmetry, each unique charge pair is represented twice in intact IgG (i.e., also K439 / D356', K370 / E357', D399 / K392' and K409 / D399' charge interactions are present in the interface). Taking advantage of this two-fold symmetry, it was demonstrated that a single charge reversion, e.g. K409D in the first chain, or D399'K in the second chain resulted in diminished homodimer formation due to repulsion of identical charges. Combining different charge reversions further enhanced this repulsive effect. It was demonstrated that expression of different CH3 domains comprising different, complementary charge reversions, could drive heterodimerisation, resulting in an increased proportion of the bispecific species in the mixture (for review see Kontermann and Brinkmann, supra; Brinkmann and Kontermann: The making of bispecific antibodies, MABS, Vol. 9, No. 2, 2017, pages 182-212, Ha et al, Frontiers in Immunology Immunoglobulin Fc Heterodimer Platform Technology: From Design to Applications in Therapeutic Antibodies and Proteins, Vol 7, article 394, 2016).

[0239] In some embodiments, the dimeric Fc is a heterodimer formed with a purity greater than about 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% when produced; or wherein the Fc is a heterodimer formed with a purity greater than about 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% when expressed or when expressed via a single cell.

[0240] The Fc region may comprise one or more modifications in at least one of the CH3 sequences. The Fc region may comprise one or more modifications in at least one of the CH2 sequences. The Fc region may comprise one or more modifications in the CH2 and in the CH3 sequences.

[0241] In some embodiments, a construct described herein comprises a heterodimeric Fc region comprising a modified CH3 domain that has been asymmetrically modified. The heterodimeric Fc region can comprise two heavy chain constant domain polypeptides: a first heavy chain polypeptide and a second heavy chain polypeptide, which can be used interchangeably provided that Fc comprises one first heavy chain polypeptide and one second heavy chain polypeptide. Generally, the first heavy chain polypeptide comprises a first CH3 sequence and the second heavy chain polypeptide comprises a second CH3 sequence.

[0242] Two CH3 sequences that comprise one or more amino acid modifications introduced in an asymmetric fashion generally results in a heterodimeric Fc region, rather than a homodimer, when the two CH3 sequences dimerize. As used herein, "asymmetric amino acid modifications" refers to any modification where an amino acid at a specific position on a first CH3 sequence is different from the amino acid on a second CH3 sequence at the same position, and the first and second CH3 sequence preferentially pair to form a heterodimer, rather than a homodimer. This heterodimerization can be a result of modification of only one of the two amino acids at the same respective amino acid position on each sequence; or modification of both amino acids on each sequence at the same respective position on each of the first and second CH3 sequences. The first and second CH3 sequence of a heterodimeric Fc region can comprise one or more than one asymmetric amino acid modification.

[0243] Another suitable method uses a platform based on BEAT® technology (Bispecific Engagement by Antibodies based on the T cell receptor) (Moretti et al, BMC Proc 7, Article Number 09 (2013)).

[0244] Additional methods for modifying monomeric Fc polypeptides to promote heterodimeric Fc formation are as described above. Therefore, the Fc region as the bispecific molecule, e.g. antibody as described herein may comprise one or more modification to promote heterodimerisation, e.g. a knob in hole (KiH) modification.

[0245] In one embodiment the Fc region comprises a KilH mutation, D265C and LALA PG Fc silencing modifications. The bispecific binding molecule may comprise an additional moiety. The additional moiety may provide further function to the molecule. For example the further moiety may be selected from a half-life extending moiety and / or a label.

[0246] The additional moiety may be selected from one or more half-life extending moieties for example, one or more PEG molecules, a liposome, a serum albumin protein an antibody or antibody fragment that binds serum albumin. The serum albumin may be human serum albumin. The bispecific binding molecule may be conjugated or linked to the additional moiety in any suitable manner.

[0247] In some embodiments, the bispecific molecule may comprise a half-life extension moiety. The inclusion of a half-life extension moiety suitably increases the half-life of the bispecific molecule when compared to the same bispecific molecule that does not include a half-life extension moiety (wherein the term “half-life” is as already defined herein).

[0248] The half-life extension moiety may comprise an antibody and / or antigen-binding fragment, a protein, and or a polypeptide. For example, the half-life extension moiety may comprise a serum albumin protein or an antibody or antigen-binding fragment that binds human serum albumin. Preferably, the half-life extension moiety may comprise an antibody or antigen-binding fragment that binds human serum albumin, more preferably an antigen-binding fragment that binds human serum albumin, more preferably a single domain antibody that binds human serum albumin, most preferably a VH domain that that binds human serum albumin.

[0249] In some embodiments, the bispecific molecule may comprise one or more further antibodies and / or antigen-binding fragments. For example, the compound may comprise a total of 3, 4, 5, etc., antibodies and / or antigen-binding fragments. In an embodiment the compound may comprise a total of 3 antibodies and / or antigen-binding fragments. The further antibodies and / or antigen-binding fragments may be any suitable antibodies and / or antigen-binding fragments. For example, the further antibodies and / or antigen-binding fragments may bind to human serum albumin or may bind to any other suitable antigen. Suitable antigens include antigens such as CD56, B7H4. It will be appreciated by the skilled person that the further antibodies and / or antigen-binding fragments may be attached to the bispecific binding molecule in any suitable manner for example recombinantly attached or chemically linked. For example production of the multispecific binding molecule of the invention may comprise recombinant expression of the molecule, wherein a nucleic acid is designed which encodes the first and / or second antigen binding domain in combination with the further antibody, antigen binding fragment or domain and the protein is expressed in a host organism such that the antigen binding domains are expressed in a linked manner. Production of the multispecific binding molecule of the invention may comprise preparing one or more of the antibodies, antigen binding fragments or antigen binding domains in a manner such that they can be chemically linked, for example by the click chemistry methods described herein. Production of the multispecific binding molecule may comprise a mixture of recombinantly expressing some of the binding domains in a linked manner and chemically linking some of the binding domains.

[0250] It will be appreciated by a person skilled in the art that in certain embodiments a further antibody or antigen-binding fragment may also be a half-life extension moiety and vice versa. For example where the further antibody or antigen-binding fragment is a serum albumin binding protein, the further antibody or antigen binding fragment would also be a half-life extension moiety.

[0251] The additional moiety may be a detectable or functional label. A label can be any molecule that produces or can be induced to produce a signal, including but not limited to fluorophores, fluorescers, radiolabels, enzymes, chemiluminescers, a nuclear magnetic resonance active label or photosensitizers. Thus, the binding may be detected and / or measured by detecting fluorescence or luminescence, radioactivity, enzyme activity or light absorbance. The molecular label may be a fluorophore. Suitable fluorophores include fluorescein isothiocyanate (FITC), phycoerythrin (PE), allophycocyanin (APC), Indocicarbocyanine (Cy5), Indocarbocyanine (Cy3), as well as those known by the trade names Alexa Fluor (such as 350, 405, 488, 532, 546, 568, 594, 647, 680, 700, 750) and DyLight (such as 405, 488, 550, 650, 680, 755, 800).

[0252] The molecular label may also be a biotin tag, derived from biotin. The labelling moiety may also be a radioisotope or a radioisotope containing moiety. Suitably, the labelling moiety is a positron emission tomography (PET) tracer. Suitable PET tracers include, for example, [18F] Fludeoxyglucose (18F) (FDG)-glucose analogue, [11 C] acetate, [11 C] methionine, [11 C] choline, copper Cu dotatate, [18F] EF5, [18F] fluciclovine, [18F] fluorocholine, [18F] fluoroethyl- L-tyrosine, [18F] fluoromisonidazole, [18F] fluorothymidine F-18, [64 Cu] Cu-ETS2, [68Ga] DOTA-pseudopeptides, [68Ga] DOTA-TATE and [68Ga] prostate-specific membrane antigen (PSMA).

[0253] The one or more further moieties may be attached to the compound at any suitable position and by any suitable means. For example, when the bispecific molecule comprises a further antibody and / or antigen-binding fragment, protein and / or polypeptide, said antibody and / or antigenbinding fragment, protein and / or polypeptide may be recombinantly attached to the bispecific binding molecule.

[0254] Suitable recombinant techniques will be known to a person skilled in the art. For example, nucleic acids encoding the bispecific molecule, and the further antibody and / or antigen-binding fragment, protein and / or polypeptide, suitably separated by a polypeptide spacer / linker sequence, may be inserted into a plasmid and expressed in a suitable expression system. Suitable expression systems include bacterial host cells, such as E. coli, CHO or other host cells expressing T7 RNA polymerase in the cell which also includes a polynucleotide encoding the bispecific molecule linked to the additional moiety that is operably linked to a T7 promoter. For example, a bacterial host cell, such as an E. coli, may include a polynucleotide encoding the T7 RNA polymerase gene operably linked to a lac promoter and expression of the polymerase and the bispecific molecule linked to the additional moiety is induced by incubation of the host cell with IPTG (isopropyl-beta-D-thiogalactopyranoside). Transformation can be by any known method for introducing polynucleotides into a host cell. Suitable transformation methods are as already described herein.

[0255] The antibody or antigen-binding fragment may be modified to increase half-life, for example by a chemical modification, especially by PEGylation, or by incorporation in a liposome, or using a serum albumin protein or an antibody or antibody fragment that binds human serum albumin. Increased half-life can also be conferred by conjugating the molecule to an antibody fragment. The term "half-life" as used herein refers to the time taken for the serum concentration of the amino acid sequence, compound or polypeptide to be reduced by 50%, in vivo, for example due to degradation of the sequence or compound and / or clearance or sequestration of the sequence or compound by natural mechanisms.

[0256] Other methods to improve half-life may comprise modifying the bispecific binding molecule. The bispecific molecule may be modified by serum albumin protein. The bispecific molecule may be conjugated to serum albumin protein. The serum albumin protein may be human serum albumin or a modified version.

[0257] The bispecific molecule may comprise an antibody or antibody fragment that binds human serum albumin. The bispecific molecule may be conjugated to a further antibody or fragment thereof that binds human serum albumin.

[0258] Increased half-life can also be conferred by conjugating the bispecific molecule to an antibody fragment, for example wherein the antibody fragment binds serum albumin. The antibody fragment that binds serum albumin may be a F(ab')2, Fab, Fab’, Fv, scFv, heavy chain, light chain, variable heavy (VH), variable light (VL) chain, CDR region, single VH or VL domain, maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, and bis-scFv, and polypeptides that contain at least a portion of an immunoglobulin that is sufficient to confer specific antigen binding to the polypeptide. In an embodiment the antigen binding fragment that binds serum albumin may be a single domain antibody for example a VH. In an embodiment the antigen binding fragment that binds serum albumin may be an scFv. The term "half-life" as used herein refers to the time taken for the serum concentration of the amino acid sequence, compound or polypeptide to be reduced by 50%, in vivo, for example due to degradation of the sequence or compound and / or clearance or sequestration of the sequence or compound by natural mechanisms.

[0259] Half-life may be increased by at least 1 .5 times, preferably at least 2 times, such as at least 5 times, for example at least 10 times or more than 20 times, greater than the half-life of the corresponding antibodies without such modification. For example, increased half-life may be more than 1 hours, preferably more than 2 hours, more preferably more than 6 hours, such as more than 12 hours, or even more than 24, 48 or 72 hours, compared to the corresponding antibodies without such modification. The in vivo half-life of the antibody or antigen-binding fragment or compound of the invention a can be determined in any manner known per se, such as by pharmacokinetic analysis. Suitable techniques will be clear to the person skilled in the art. Half-life can for example be expressed using parameters such as the t1 / 2-alpha t1 / 2-beta and the area underthe curve (AUC). It will be appreciated by a person skilled in the art that reference to the half-life of an antibody may also refer to the half-life of the compounds of the invention (and may be used interchangeably herein).

[0260] As described herein the bispecific molecule may comprise a first and second antibody, antigen binding fragment, or antigen binding domain, in such embodiment the first and second may be attached or linker together. The first and second antibody, antigen binding fragment, or antigen binding domain may be attached via a linker. The linker may be formed when the first and second antibody, antigen binding fragment, or antigen binding domain are conjugated together to form a bispecific molecule. The first and second antibody, antigen binding fragment, or antigen binding domain may be conjugated together via click chemistry.

[0261] In order to conjugate the first and second antibody, antigen binding fragment, or antigen binding domain together a thiol containing group may be introduced into the first and or second antibody, antigen binding fragment, or antigen binding domain In order to conjugate the first and second antibody, antigen binding fragment, or antigen binding domain together cysteine residues may be introduced into the first and or second antibody, antigen binding fragment, or antigen binding domain. These cysteine residues may be referred to herein a non-naturally occurring cysteine residues i.e., the cysteine residue has been recombinantly introduced, for example the wild-type residue has been replaced with a cysteine using site directed mutagenesis. A “non-naturally occurring cysteine residue” refers to a cysteine residue that has been introduced at a position in the polypeptide wherein a different amino acid was present in the wild-type sequence. Methods for introducing cysteine resides are known in the art for example site directed mutagenesis. The first and second antibody or antigen-binding fragment each independently comprise a substitution to a cysteine residue at one of more of positions 153, 156, 168, 173 and / or 207 of the CH1 domain according to Kabat numbering. Thus, the first and second antibody and antigenbinding fragment each independently comprise one or more non-naturally occurring cysteine residue(s). In an embodiment the first and second antibody or antigen-binding fragment each independently comprise a substitution to a cysteine residue at position 207 according to Kabat numbering. Position 207 of the CH1 domain according to Kabat numbering corresponds to position 103 of the CH1 domain according to IMGT unique numbering for C-domain and position 82 according to IMGT exon numbering.

[0262] Position 153 of the CH1 domain according to Kabat numbering corresponds to position 38 of the CH1 domain according to IMGT unique numbering for C-domain and position 38 according to IMGT exon numbering. Position 156 of the CH1 domain according to Kabat numbering corresponds to position 40 of the CH1 domain according to IMGT unique numbering for C- domain and position 40 according to IMGT exon numbering. Position 168 of the CH1 domain according to Kabat numbering corresponds to position 45.3 of the CH1 domain according to IMGT unique numbering for C-domain and position 48 according to IMGT exon numbering. Position 173 of the CH1 domain according to Kabat numbering corresponds to position 80 of the CH1 domain according to IMGT unique numbering for C-domain and position 52 according to IMGT exon numbering.

[0263] The first antibody or antigen-binding fragment may comprise a substitution to a cysteine residue at position 207 of the CH1 domain, such as an I207C substitution, according to Kabat numbering. The second antibody or antigen-binding fragment may comprise a substitution to a cysteine residue at position 207 of the CH1 domain, such as an I207C substitution, according to Kabat numbering. In an embodiment the first and / or second antibody or antigen-binding fragment may comprise a substitution to a cysteine residue at position 207, such as an I207C substitution, according to Kabat numbering. In an embodiment the first antibody or antigen-binding fragment comprises an antigen binding domain that binds to CD7 and comprises a substitution to a cysteine residue at position 207, such as an I207C substitution, according to Kabat numbering wherein the heavy chain comprises a sequence selected from SEQ ID NO. 22, SEQ ID NO. 26, SEQ ID NO. 30, SEQ ID NO. 34, or SEQ ID NO. 38.

[0264] The bispecific antibody or antigen binding fragment comprises a first sulphur atom, S’, wherein the first sulphur atom is derived from a non-naturally occurring cysteine residue of the first antibody or antigen-binding fragment. The bispecific antibody or antigen binding fragment comprises a second sulphur atom, S”, wherein the second sulphur atom is derived from a non- naturally occurring cysteine residue of the second antibody or antigen-binding fragment. It will be appreciated by the skilled person that the first and second sulphur atoms are suitably derived from a substituted cysteine residue (i.e., non-naturally occurring cysteine residue) at one of positions 153, 156, 168, 173 and / or 207, according to Kabat numbering, on the first and second antibody or antigen-binding fragment, respectively.

[0265] The first sulphur atom, S’, may be derived from a non-naturally occurring cysteine at position 207 of the CH1 domain of the first antibody or antigen-binding fragment according to Kabat numbering. The second sulphur atom, S”, may be derived from a non-naturally occurring cysteine at position 207 of the second antibody of antigen-binding fragment according to Kabat numbering. For the avoidance of doubt, by “derived from”, and like terms as used herein, in relation to the sulphur atoms is meant that said sulphur atom is that present in the side chain of a cysteine residue, (i.e.: -N-CH(CH3SH)-C(=O)-).

[0266] The linker which attaches the first and second antibody, antigen-binding fragment, antigen binding domain is attached to each of the first and second sulphur atoms, S’ and S”, of the first and second antibody or antigen-binding fragment, respectively.

[0267] The linker may be any suitable linker.

[0268] The linker may comprise a succinimide group. For example, the linker may comprise a succinimide group attached to the first and / or second sulphur atom. Preferably, the linker may comprise a succinimide group attached to each of the first and second sulphur atom. The succinimide group(s) may suitably be derived from the reaction of the first and / or second sulphur atom with a maleimide group.

[0269] The linker may comprise one or more polyethylene glycol (PEG) groups. For example, the linker may comprise 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, etc. polyethylene glycol groups. As such, the linker may comprise at least one moiety of the formula -(-O-CH2- CH2-)n-, wherein n is from 1 to 20, such as from 1 to 15, such as from 1 to 10, such as from 1 to 5, such as from 1 to 3, or even 3. Preferably, the linker may comprise two moieties of the formula -(-O-CH2-CH2-)n-, wherein n is from 1 to 20, such as from 1 to 15, such as from 1 to 10, such as from 1 to 5, such as from 1 to 3, or even 3, wherein each moiety of the formula -(- O-CH2-CH2-)n- is separated by a suitable chemical moiety.

[0270] The linker may comprise a polycyclic group. By “polycyclic”, and like terms as used herein, is meant a group comprising a plurality of ring moieties wherein at least two of said ring moieties share at least one atom, preferably at least two atoms, in common. Preferably, the polycyclic group comprises first and second fused rings that share at least one atom in common, preferably at least two atoms in common, more preferably two atoms in common. In an embodiment, the polycyclic group may comprise a first 6-membered heterocyclic ring fused to a second 8- membered ring, wherein the first and second rings share two atoms in common.

[0271] As defined herein, the linker may be formed via a click chemistry reaction. The term “click chemistry reaction” refers to a group of reactions that are modular lining reactions, there are four key classes of click reactions including cycloadditions, nucleophilic ring openings, non-aldol carbonyl chemistry and carbon multiple bond additions. Thus, preferably, the polycyclic group is formed via a click chemistry reaction, suitably wherein a first ring is caused to fuse with a second ring via a click chemistry reaction. For example, the polycyclic group may be derived from the click chemistry reaction of a cyclic alkene or cyclic alkyne group with a tetrazine group, preferably a cyclooctene group or bicyclononyne group with a tetrazine group, most preferably transcyclooctene or bicyclo[6.1 ,0]non-4-yne with a tetrazine group.

[0272] The bispecific binding molecule may comprise a first and second antigen binding domain wherein the first antigen binding domain comprises i) a heavy chain variable (VH) region comprising SEQ ID NO. 21 or a sequence comprising at least 90%, 92%, 95%, 97% or 99% sequence identity thereto, and ii) a light chain variable (VL) region comprising SEQ ID NO. 23 or a sequence comprising at least 80%, 85%, 90%, 92%, 95%, 97% or 99% sequence identity thereto, and the second antigen binding domain comprises i) a heavy chain variable (VH) region comprising a HC CDR1 comprising SEQ ID NO. 2, a HC CDR2 comprising SEQ ID NO. 3, a HC CDR3 comprising SEQ ID NO. 4, and ii) a light chain variable (VL) region comprising a LC CDR1 comprising SEQ ID NO. 7, a LC CDR2 comprising SEQ ID NO. 8, a LC CDR3 comprising SEQ ID NO. 9 wherein the first and second antigen binding domains comprise a cysteine residue at one of more of positions 153, 156, 168, 173 and / or 207 of the CH1 domain according to Kabat numbering.

[0273] The bispecific binding molecule may comprise a first and second antigen binding domain wherein the first antigen binding domain comprises i) a heavy chain variable (VH) region comprising SEQ ID NO. 21 or a sequence comprising at least 90%, 92%, 95%, 97% or 99% sequence identity thereto, and ii) a light chain variable (VL) region comprising SEQ ID NO. 23 or a sequence comprising at least 80%, 85%, 90%, 92%, 95%, 97% or 99% sequence identity thereto, and the second antigen binding domain comprises i) a heavy chain variable (VH) region comprising SEQ ID NO. 1 and ii) a light chain variable (VL) region comprising SEQ ID NO. 6 wherein the first and second antigen binding domains comprise a cysteine residue at one of more of positions 153, 156, 168, 173 and / or 207 of the CH1 domain according to Kabat numbering.

[0274] The bispecific binding molecule may comprise a first and second antigen binding domain wherein the first antigen binding domain comprises i) a heavy chain variable (VH) region comprising SEQ ID NO. 21 or a sequence comprising at least 90%, 92%, 95%, 97% or 99% sequence identity thereto, and ii) a light chain variable (VL) region comprising SEQ ID NO. 23 or a sequence comprising at least 80%, 85%, 90%, 92%, 95%, 97% or 99% sequence identity thereto, and the second antigen binding domain comprises i) a heavy chain variable (VH) region comprising SEQ ID NO. 1 and ii) a light chain variable (VL) region comprising SEQ ID NO. 6 wherein the first and second antigen binding domains comprise a cysteine residue at one of more of positions 153, 156, 168, 173 and / or 207 of the CH1 domain according to Kabat numbering and wherein the first and second antigen binding domains are conjugated together via a linker which attaches the first and second antigen-binding domain, wherein the linker is attached to each of the first and second sulphur atoms, S’ and S”, of the first and second antigenbinding domain, respectively.

[0275] The bispecific binding molecule may comprise a first and second antigen binding domain wherein the first antigen binding domain comprises i) a heavy chain variable (VH) region comprising SEQ ID NO. 21 or a sequence comprising at least 90%, 92%, 95%, 97% or 99% sequence identity thereto, and ii) a light chain variable (VL) region comprising SEQ ID NO. 23 or a sequence comprising at least 80%, 85%, 90%, 92%, 95%, 97% or 99% sequence identity thereto, and the second antigen binding domain comprises i) a heavy chain variable (VH) region comprising SEQ ID NO. 1 and ii) a light chain variable (VL) region comprising SEQ ID NO. 6 wherein the first and second antigen binding domains comprise a cysteine residue at one of more of positions 153, 156, 168, 173 and / or 207 of the CH1 domain according to Kabat numbering and wherein the first and second antigen binding domains are conjugated together via a linker which attaches the first and second antibody, antigen-binding fragment, antigen binding domain is attached to each of the first and second sulphur atoms, S’ and S”, of the first and second antibody or antigen-binding fragment, respectively and wherein the linker is formed via a click chemistry reaction.

[0276] The first and second antibody, antigen-binding fragment or antigen binding domain are conjugated together via click chemistry. The terms “conjugation” and “conjugate(d)” refer to chemical linkages, either covalent or non-covalent, which proximally associates one molecule of interest with a second molecule of interest.

[0277] As such, each of the first and second antibody, antigen-binding fragment or antigen binding domain typically comprise at least one functional group capable of participating in a click chemistry reaction. As used herein, the functional group capable of participating in a click chemistry reaction may be present as part of a “linking arm” or a “click handle”, “click arm”, etc (which terms may be used interchangeably herein). Preferably, the first and second antibody or antigen binding fragment or antigen binding domain may each comprise a click handle, wherein each click handle comprises a complimentary conjugating group, such as those defined herein.

[0278] A click handle may be attached to the first and / or second antibody or antigen binding fragment or antigen binding domain, as appropriate, in any suitable manner. For example, a click handle may be attached to the first and / or second antibody or antigen-binding fragment, as appropriate, via the reaction of a thiol group of one or more of the non-naturally occurring cysteine residues as described herein and a maleimide, bromomaleimide, bromopyridazinedione, chloroacetamide, bromoacetamide, iodoacetamide, a-haloketone, unsaturated carbonyl, vinylsulfone, vinyl-pyridine, vinyl-pyridinium and / or unsaturated phosphonamidite group (Chem. Sci., 2021 , 12, 9060-9068). Suitably, before attachment, a click handle may comprise a maleimide, bromomaleimide bromopyridazinedione, a-haloketone, unsaturated carbonyl, vinylsulfone, vinyl-pyridine, vinyl-pyridinium and / or unsaturated phosphonamidite group and a conjugating group, wherein the conjugating group is operable to participate in a click chemistry reaction.

[0279] Any suitable click chemistry may be used to conjugate together the first and second antibody, antigen-binding fragment, or antigen binding domain. Suitable click chemistries will be known to a person skilled in the art. It will be appreciated by a person skilled in the art that the choice of click chemistry will typically depend upon the functional groups that are to be conjugated together. The click chemistry may be selected from a thiol-ene reaction, a copper catalyzed azide-alkyne cycloaddition (CuAAC) reaction, a strained-promoted azide-alkyne click chemistry (SPAAC) reaction and / or an inverse electron demand Diels-Alder (iEDDA) reaction. Preferably, the click chemistry may be an inverse electron demand Diels-Alder (iEDDA) reaction.

[0280] A thiol-ene reaction (which may also be referred to as an alkene hydrothiolation reaction) is the reaction between a thiol group, i.e., R-SH, and an alkene to form a thioether. The reaction results in an anti-Markovnikov addition of a thiol compound to an alkene. Thiol-ene reactions may proceed through two mechanisms: free-radical additions and catalyzed Michael additions. Free- radical addition reactions can be initiated by light, heat or radical initiators, which form a thiyl radical species. The radical then propagates with an ene functional group via an anti- Markovnikov addition to form a carbon-centred radical. A chain-transfer step removes a hydrogen radical from a thiol, which can subsequently participate in multiple propagation steps. Michael addition reactions are catalyzed by either a base or a nucleophile, resulting in a similar anti-Markovnikov addition product as the free-radical addition reactions. A thiol-ene reaction is exemplified in scheme I.

[0281] Scheme I: thiol-ene reaction (maleimide)

[0282] In some embodiments, the reaction may be between a thiol group and a maleimide group. As such, the first and second antibody, antigen-binding fragment, or antigen binding domain may be conjugated together via a thiol-maleimide reaction.

[0283] In some embodiments, the first antibody, antigen-binding fragment or antigen binding domain may be conjugated to the second antibody or antigen-binding fragment via the -S’-H group present on the first antibody, antigen-binding fragment, or antigen binding domain before conjugation and an alkene group, such as a cyclic alkene group, or even a maleimide group, present on the second antibody or antigen-binding fragment before conjugation or vice versa. In such embodiments, the second antibody or antigen-binding fragment (or first antibody or antigen-binding fragment if vice versa) may comprise a linker arm, L” (or L’), comprising an alkene group, such as a cyclic alkene group, or even a maleimide group. Preferably, the alkene group, such as cyclic alkene group, or even maleimide group, is terminal. The linker arm may itself be attached to the first or second antibody or antigen-binding fragment, as appropriate, via click chemistry, preferably via a thiol-ene reaction, more preferably via a thiol-maleimide reaction.

[0284] In other embodiments, the -S’-H and S”-H groups as present on the first and second antibody, antigen-binding fragment or antigen binding domain before conjugation, respectively, may be reacted with a linker unit of formula X’-L2-X”, wherein L2is a linker and each of X’ are alkene groups, such as cyclic alkene groups, or even maleimide groups.

[0285] A copper catalyzed azide-alkyne cycloaddition (CuAAC) reaction is the reaction between an azide and an alkyne to form a 1 ,5-disubstituted 1 ,2,3-triazole. The reaction between azide and alkyne is very selective. The reaction is also quick and pH sensitive. A CuAAC reaction is exemplified in scheme II:

[0286] Scheme II: CuAAC reaction

[0287] The catalyst for the CuAAC reaction may comprise a copper(l) catalyst, such as cuprous bromide or cuprous iodide and / or a mixture of copper(ll) and a reducing agent, such as sodium ascorbate, to produce copper(l) in situ (i.e., in the reaction mixture). Alternatively, a copper-free catalyst may be used. For example, a pentamethylcyclopentadienyl ruthenium chloride complex may be used as a catalyst to catalyse the azide-alkyne cycloaddition.

[0288] A strained-promoted azide-alkyne click chemistry (SPAAC) reaction is the reaction between an azide and a strained alkyne. The strained alkyne may comprise a strained cyclooctyne, such as difluorooctyne (DIFO), dibenzylcyclooctyne (DIBO) and biarylazacyclooctynone (BARAC). When DIFO is used, for example, the electron-withdrawing, propargylic, gem-fluorines act together with the ring strain to destabilize the alkyne. This destabilization increases the reaction driving force, and the desire of the cycloalkyne to relieve its ring strain. Substituents other than fluorines on the cyclooctyne, such as benzene rings for example, also produce this effect. SPAACs reactions are exemplified in schemes Illa and lllb.

[0289] Scheme lllb: SPAAC reaction (DIBO)

[0290] An inverse electron demand Diels-Alder (iEDDA) reaction is the reaction between an electron poor diene group, such as a tetrazine group, and an electron rich dienophile group, such as a cycloalkene or cycloalkyne group, for example a cyclooctene or bicyclononyne group. Frontier orbital overlap between the HOMO(Dienophiie) and LUMO(Diene) is the driving force for this reaction. High ring strain of dienophiles increases the HOMO energy leading to a small HOMO(Dienophiie)- LUMO(Diene) separation. Thus, three-membered and four-membered cycloalkenes or cycloalkynes, as well as trans-cycloalkenes, make ideal dienophile substrates due to their high ring strain. Electron-donating substituents on the dienophile and electron-withdrawing substituents on the diene accelerate the iEDDA reaction by further reducing the HOMO(Dienophiie)- LUMO(Diene) separation. An iEDDA reaction is exemplified in scheme IV:

[0291] Scheme IV: iEDDA reaction

[0292] Preferably, the click chemistry may be an inverse electron demand Diels-Alder (iEDDA) reaction. As such, preferably, the functional groups to be conjugated together may suitably be a tetrazine group and a cycloalkene group, such as a cyclooctene group, and / or a cycloalkyne group, such as a cyclooctyne group. In some embodiments, the functional groups to be conjugated together may be a tetrazine group and a cyclooctene group, such as trans-cyclooctene (TCO). In some embodiments, the functional groups to be conjugated together may be a tetrazine group and a cyclooctyne group, such as bicyclononyne (BCN). It will be appreciated by a person skilled in the art that bicyclononyne includes an 8-membered ring having an unsaturated carbon-carbon triple bond, i.e., a cyclooctyne group, and a further s membered ring (sharing 2 carbon atoms in common with the cyclooctyne group).

[0293] In some embodiments, the first and second antibody, antigen-binding fragments or antigen binding domain may be conjugated together via a click chemistry reaction between a first linker arm, L’, attached to the first antibody or antigen-binding fragment or antigen binding domain and a second linker arm, L”, attached to the second antibody or antigen-binding fragment. Suitably, the first linker arm, L’, may be attached to the first antibody or antigen-binding fragment via the first sulphur atom, S’. Suitably, the second linker arm, L”, may be attached to the second antibody or antigen-binding fragment via the second sulphur atom, S”.

[0294] Before the first and second antibody, antigen-binding fragment or antigen binding domain are conjugated together, the first linker arm, L’, may comprise a conjugating group selected from an azide, nitrone, tetrazine, tetrazole, aliphatic or cyclic alkene or aliphatic or cyclic alkyne group. Before the first and second antibody or antigen-binding fragment are conjugated together, the second linker arm, L”, may comprise a conjugating group selected from an azide, nitrone, tetrazine, tetrazole, aliphatic or cyclic alkene and / or aliphatic or cyclic alkyne group, with the proviso that when the conjugating group of the first linker arm, L’, is an azide, nitrone, tetrazine or tetrazole group, then the conjugating group of the second linker arm, L”, is an aliphatic or cyclic alkene or aliphatic or cyclic alkyne group, and when the conjugating group of the first linker arm, L’, is an aliphatic or cyclic alkene or aliphatic or cyclic alkyne group, then the conjugating group of the second linker arm, L”. is an azide, nitrone, tetrazine or tetrazole group.

[0295] In some embodiments, before the first and second antibody or antigen-binding fragment are conjugated together, the first linker arm, L’, may comprise a tetrazine group. In some embodiments, before the first and second antibody or antigen-binding fragment are conjugated together, the first linker arm, L’, may comprise a tetrazine group and the second linker arm, L”, may comprise an aliphatic or cyclic alkene or an aliphatic or cyclic alkyne group, such as a cyclic alkene group or a cyclic alkyne group. In some embodiments, before the first and second antibody or antigen-binding fragment are conjugated together, the first linker arm, L’, may comprise a tetrazine group. In some embodiments, before the first and second antibody or antigen-binding fragment are conjugated together, the first linker arm, L’, may comprise a tetrazine group and the second linker arm, L”, may comprise a cyclooctene group, such as transcyclooctene (TCO), or a cyclooctyne group, such as bicyclononyne (BCN).

[0296] For the avoidance of doubt, tetrazine as used herein is intended to include 1 ,2,3,4-tetrazine, 1 , 2, 3, 5-tetrazine, and 1 , 2, 4, 5-tetrazine, as well as derivatives thereof, such as 6-methyl tetrazine.

[0297] For the avoidance of doubt, tetrazole as used herein is intended to include 1 / 7-tetrazole, 2 / 7- tetrazole and 5 / 7-tetrazole, as well as derivatives thereof, such as 5-methy I- 1 / 7-tetrazole and 1- methy I- 1 / 7-tetrazole

[0298] The bispecific molecule may comprise a first antigen binding domain having a binding affinity (KD) to CD7 of between 5 x10-10M and 2 x10-7M, between 6 x10-10M and 2 x10-7M, between 8 x10-10M and 2 x10-7M, between 9 x10-10M and 2 x10-7M, between 1 x10-9M and 2 x10-7M, between 2 x10-9M and 2 x10-7M, between 3 x10-9M and 2 x10-7M, between 4 x10-9M and 2 x10-7M, between 5 x10-9M and 2 x10-7M, between 6 x10-9M and 2 x10-7M, between 8 x10-9M and 2 x10-7M, between 9 x10-9M and 2 x10-7M, between 1 x10-8M and 2 x10-7M, between 2 x10-8M and 2 x10-7M, between 3 x10-8M and 2 x10-7M, between 4 x10-8M and 2 x10-7M, between 5 x10-10M and 2 x10-8M, between 6 x10-10M and 2 x10-8M, between 8 x10-10M and 2 x1 O'8M, between 9 x10-10M and 2 x10-8M, between 5 x10-9M and 2 x10-8M, between 6 x10-9M and 2 x10-8M, between 8 x10-9M and 2 x10-8M, between 9 x10-9M and 2 x10-8M, between 5 x10-9M and 2 x10-9M’ The bispecific molecule may comprise a second antigen binding domain has a binding affinity (KD) to CD33 of between 1 .5 x1 O’10M and 3 x1 O’8M, between 2 x1 O’10M and 4 x1 O’8M, between 3 x10-10M and 5 x1 O’8M, between 4 x1 O’10M and 6 x1 O’8M, between 5 x1 O’10M and 7 x1 O’8M, between 6 x1 O’10M and 8 x10-8M, between 7 x1 O’10M and 9 x10-8M, between 8 x1 O’10M and 1 x1 O'9M, between 1 .5 x1 O’9M and 3 x1 O’8M, between 2 x1 O’9M and 4 x1 O’8M, between 3 x1 O’9M and 5 x1 O’8M, between 4 x1 O’9M and 6 x1 O’8M, between 5 x1 O’9M and 7 x1 O’8M, between 6 x10-9M and 8 x10-8M, between 7 x10-9M and 9 x10-8M, between 8 x10-9M and 1 x10-8M, between 1 .5 x1 O-10M and 3 x10-9M, between 1 .5 x10-9M and 3 x10-9M.

[0299] The bispecific molecule is capable of binding both CD7 and CD33, upon binding to the target antigens the bispecific molecule may induce receptor mediate internalisation. The bispecific molecule is capable of inducing CD33 and / or CD7 receptor mediated internalization into a CD33+ and / or CD7+ cell. The bispecific molecule is capable of inducing CD33 receptor mediated internalization into a CD33+ cell. The bispecific molecule is capable of inducing CD7 receptor mediated internalization into a CD7+ cell. The bispecific molecule is capable of inducing CD33 receptor mediated internalization into a CD33+CD7+ cell. The bispecific molecule is capable of inducing CD7 receptor mediated internalization into a CD33+CD7+ cell.

[0300] The inventors show herein that the bispecific molecules are capable of selectively targeting CD7+ CD33+ dual positive cells, providing excellent cell specificity. The bispecific molecule is capable of selectively targeting cells expressing CD7+ and CD33+, compared to cells expressing CD7+ or cells expressing CD33+. As such the bispecific molecule has reduced off- target effects which may occur as a result of targeting single positive cells i.e., cells expressing CD7+ or cells expressing CD33+.

[0301] Dual positive cells expressing both CD7+ CD33+ may be found in malignant cells. The bispecific binding molecule may be capable of bispecifically binding to a cell expressing CD7+ and CD33+, wherein the cell is a malignant cell.

[0302] Immunoconjugate

[0303] The bispecific binding molecule of the invention may be conjugated to a payload. A “payload” may be selected from but not limited to a cell killing agent, an immune-modulating payload, a macrophage class switching agent, a detectable label or a light activatable payload. In certain embodiments where the bispecific binding molecule is conjugated to a payload this is referred to as an immunoconjugate.

[0304] In one embodiment, the payload may be a cell killing agent. In one embodiment, the payload may be a macrophage class switching agent. In one embodiment, the payload may be an immune-modulating payload. In one embodiment, the payload may be a light activatable payload. In one embodiment, the payload may be a molecular label. By “molecular label”, and like terms as used herein, is meant a group that is operable to aid the detection of the compound. Detection of the compound may be ex vivo and / or in vivo. Examples of suitable molecular labels include, but are not limited to, fluorescent molecules, p-galactosidase, luciferase molecules, chemical dyes, fluorophores and / or radioisotopes.

[0305] The additional moiety may be a detectable or functional label. A label can be any molecule that produces or can be induced to produce a signal, including but not limited to fluorophores, fluorescers, radiolabels, enzymes, chemiluminescers, a nuclear magnetic resonance active label or photosensitizers. Thus, the binding may be detected and / or measured by detecting fluorescence or luminescence, radioactivity, enzyme activity or light absorbance. The molecular label may be a fluorophore. Suitable fluorophores include fluorescein isothiocyanate (FITC), phycoerythrin (PE), allophycocyanin (APC), Indocicarbocyanine (Cy5), Indocarbocyanine (Cy3), as well as those known by the trade names Alexa Fluor (such as 350, 405, 488, 532, 546, 568, 594, 647, 680, 700, 750) and DyLight (such as 405, 488, 550, 650, 680, 755, 800). The molecular label may also be a biotin tag, derived from biotin. The labelling moiety may also be a radioisotope or a radioisotope containing moiety. Suitably, the labelling moiety is a positron emission tomography (PET) tracer. Suitable PET tracers include, for example, [18F] Fludeoxyglucose (18F) (FDG)-glucose analogue, [11 C] acetate, [11 C] methionine, [11 C] choline, copper Cu dotatate, [18F] EF5, [18F] fluciclovine, [18F] fluorocholine, [18F] fluoroethyl- L-tyrosine, [18F] fluoromisonidazole, [18F] fluorothymidine F-18, [64 Cu] Cu-ETS2, [68Ga] DOTA-pseudopeptides, [68Ga] DOTA-TATE and [68Ga] prostate-specific membrane antigen (PSMA).

[0306] As used herein, an immune-modulating payload includes any moiety that modulates the immune system, for example which stimulates the immune system and / or kills the target cell. Thus, a moiety that has immuno-activating and / or antineoplastic activities can be used. Such moieties may be synthetic peptides that recognise the specific target and trigger (agonist) or block (antagonist) inflammatory responses. The target may be a pattern recognition receptor (PRR), including Toll-like receptors (TLRs), NOD-like receptors (NLRs), RIG-l-like receptors (RLRs), C- type lectin receptors (CLRs) and cytosolic dsDNA sensors (CDSs).

[0307] Examples of payloads include agonists for the stimulator of interferon genes protein (STING; transmembrane protein 173; TMEM173). Such payloads include cyclic dinucleotides and compounds listed in see WO2021113679). Activation of the STING pathway triggers an immune response that results in generation of specific killer T-cells that shrink tumours and can provide long-lasting immunity so the tumours do not recur. Alternatively, payloads that act on toll-like receptors (TLRs) may be used. For example, agonists that bind to TLR7 and / or TLR8 can be used. Another example is a macrophage class switching agent.

[0308] The payload may be a cytotoxic payload or a therapeutic compound, peptide or polypeptide. In particular, the payload is preferably a cytotoxin.

[0309] Preferably the cytotoxin is a biologically active cytotoxic material. The cytotoxin may be selected from the group comprising auristatins, maytansinoids, tubulysins, RNA polymerase II inhibitors, transcription inhibitors, calicheamicins, duocarmycins, pyrrolobenzodiazepines (in particular pyrrolobenzodiazepine dimers), camptothecin analogues, topoisomerase inhibitors and doxorubicin.

[0310] However, additionally or alternatively, the cytotoxin could also be selected from other known cytotoxins including ricin subunits and other peptide based cytotoxic materials.

[0311] In some embodiments the payload may be a cytotoxic or cytostatic agent, i.e., a compound that kills or inhibits tumour cells. Such agents may impart their cytotoxic and cytostatic effects by mechanisms including tubulin binding, DNA binding, proteasome and / or topoisomerase inhibition. The term "cytotoxic agent" as used herein refers to a substance that inhibits or prevents the function of cells and / or causes destruction of cells. The term is intended to include radioactive isotopes (e.g., <211 >At, <131 >l, <125>1 , <90>Y, <186>Re, <188>Re, <153>Sm, <212>Bi, <32>P, <60>C, and radioactive isotopes of Lu), chemotherapeutic agents, and toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including synthetic analogs and derivatives thereof. A "chemotherapeutic agent" and "anticancer agent" are terms that denote a chemical compound useful in the treatment of cancer, and which may be administered in combination therapy with the antibody drug conjugate compounds of the invention. Examples of chemotherapeutic agents include Erlotinib (TARCEV A(R), Genentech / OSI Pharm.), Bortezomib (VELCADE(R), Millenium Pharm.), Fulvestrant (FASLODEX(R), Astrazeneca), Sutent (SUI 1248, Pfizer), Letrozole (FEMARA(R), Novartis), Imatinib mesylate (GLEEVEC(R), Novartis), PTK787 / ZK 222584 (Novartis), Oxaliplatin (Eloxatin(R), Sanofi), 5-FU (5-fluorouracil), Leucovorin, Rapamycin (Sirolimus, RAPAMUNE(R), Wyeth), Lapatinib (GSK572016, GlaxoSmithKline), Lonafarnib (SCH 66336), Sorafenib (BAY43- 9006, Bayer Labs.), and Gefitinib (IRESSA(R), Astrazeneca), AG1478, AG1571 (SU 5271 ; Sugen), alkylating agents such as thiotepa and CYTOXAN(R) cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylolomelamine; TLK 286 (TELCYTA(TM)); acetogenins (especially bullatacin and bullatacinone); delta-9-tetrahydrocannabinol (dronabinol, MARINOL(R)); beta-lapachone; lapachol; colchicines; betulinic acid; a camptothecin (including the synthetic analogue topotecan (HYCAMTIN(R)), CPT-II (irinotecan, CAMPTOSAR(R)), acetylcamptothecin, scopolectin, and 9- aminocamptothecin); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); podophyllotoxin; podophyllinic acid; teniposide; cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CBI-TMI); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; bisphosphonates, such as clodronate; antibiotics such as the enediyne antibiotics (e. g., calicheamicin, especially calicheamicin gammall and calicheamicin omegall (see, e.g., Agnew, Chem Inti. Ed. Engl, 33: 183-186 (1994)) and anthracyclines such as annamycin, AD 32, alcarubicin, daunorubicin, dexrazoxane, DX-52-1 , epirubicin, GPX- 100, idarubicin, KRN5500, menogaril, dynemicin, including dynemicin A, an esperamicin, neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores, aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN(R) doxorubicin (including morpholino- doxorubicin, cyanomo[phi]holino-doxorubicin, 2-pyrrolino-doxorubicin, liposomal doxorubicin, and deoxydoxorubicin), esorubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, and zorubicin; folic acid analogues such as denopterin, pteropterin, and trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, and testolactone; anti-adrenals such as aminoglutethimide, mitotane, and trilostane; folic acid replenisher such as folinic acid (leucovorin); aceglatone; anti-folate anti- neoplastic agents such as ALEMTA(R), LY231514 pemetrexed, dihydrofolate reductase inhibitors such as methotrexate, antimetabolites such as 5-fluorouracil (5-FU) and its prodrugs such as UFT, S-l and capecitabine, and thymidylate synthase inhibitors and glycinamide ribonucleotide formyltransferase inhibitors such as raltitrexed (TOMUDEX<1 >A, TDX); inhibitors of dihydropyrimidine dehydrogenase such as eniluracil; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfornithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; 2-ethylhydrazide; procarbazine; PSK(R) polysaccharide complex (JHS Natural Products, Eugene, OR); razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine (ELDISENE(R), FILDESIN (R)); dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids and taxanes, e.g., TAXOL(R) paclitaxel (Bristol-Myers Squibb Oncology, Princeton, NJ.), ABRAXANE(TM) Cremophor-free, albumin-engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, Illinois), and TAXOTERE(R) doxetaxel (Rh[delta]ne-Poulenc Rorer, Antony, France); chloranbucil; gemcitabine (GEMZAR(R)); 6-thioguanine; mercaptopurine; platinum; platinum analogs or platinum-based analogs such as cisplatin, oxaliplatin and carboplatin; vinblastine (VELBAN(R)); etoposide (VP- 16); ifosfamide; mitoxantrone; vincristine (ONCOVIN(R)); vinca alkaloid; vinorelbine (NAVELBINE(R)); novantrone; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; topoisomerase inhibitor RFS 2000; topoisomerase inhibitor Dxd, difluorometlhylornithine (DMFO); retinoids such as retinoic acid; pharmaceutically acceptable salts, acids or derivatives of any of the above; as well as combinations of two or more of the above such as CHOP, an abbreviation for a combined therapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone, and FOLFOX, an abbreviation for a treatment regimen with oxaliplatin (ELOXATESf(TM)) combined with 5-FU and leucovorin.

[0312] Also encompassed by the term “cytotoxic agent” are anti-hormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including NOLVADEX(R) tamoxifen), raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LYI 17018, onapristone, and FARESTON(R) toremifene; aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as, for example, 4(5)-imidazoles, aminoglutethimide, MEGASE(R) megestrol acetate, AROMASIN(R) exemestane, formestanie, fadrozole, RIVISOR(R) vorozole, FEMARA(R) letrozole, and ARHVIIDEX(R) anastrozole; and anti-androgens such as fiutamide, nilutamide, bicalutamide, leuprolide, and goserelin; as well as troxacitabine (a 1 ,3-dioxolane nucleoside cytosine analog); antisense oligonucleotides, particularly those that inhibit expression of genes in signaling pathways implicated in abherant cell proliferation, such as, for example, PKC-alpha, Raf, H-Ras, and epidermal growth factor receptor (EGF-R); vaccines such as gene therapy vaccines, for example, ALLOVECTIN(R) vaccine, LEUVECTIN(R) vaccine, and VAXID(R) vaccine; PROLEUKIN(R) rlL-2; LURTOTECAN(R) topoisomerase 1 inhibitor; ABARELIX(R) rmRH; and pharmaceutically acceptable salts, acids or derivatives of any of the above

[0313] The cytotoxic or cytostatic agent may be, for example, a peptide toxin, a small molecule toxin or a radioisotope. This is also referred to herein as drug or cytotoxic payload. The bispecific molecule may be provided as an antibody drug conjugate (ADC). The ADC comprises the bispecific binding molecule of the invention conjugated to a drug also referred to herein as a “payload”.

[0314] In one embodiment the cytotoxic or cytostatic agent may be a tubulin inhibitor; or a DNA interacting agent. Tubulin inhibitors modulate tubulin polymerisation. DNA interacting agents target cellular DNA.

[0315] In an embodiment the cytotoxic or cytostatic agent is a tubulin inhibitor. In an embodiment, the tubulin inhibitor is selected from the group comprising of: (a) an auristatin; and (b) a maytansine derivative. In an embodiment, the cytotoxic or cytostatic agent is an auristatin. Auristatins include synthetic derivatives of the naturally occurring compound Dolastatin-10. Auristatins are a family of antineoplastic I cytostatic pseudopeptides. Dolastatins are structurally unique due to the incorporation of 4 unusual amino acids (Dolavaine, Dolaisoleuine, Dolaproine and Dolaphenine) identified in the natural biosynthetic product. In addition, this class of natural product has numerous asymmetric centres defined by total synthesis studies by Pettit et al (US 4,978,744). It would appear from structure activity relationships that the Dolaisoleuine and Dolaproine residues appear necessary for antineoplastic activity (US 5,635,483 and US 5,780,588). In an embodiment, the auristatin is selected from the group consisting of: Auristatin E (AE); Monomethylauristatin E (MMAE); Auristatin F (AF), monomethylauristatin F (MMAF); vcMMAE; vcMMAF; mcMMAE and mcMMAF. In an embodiment, the cytotoxic or cytostatic agent is a maytansine or a structural analogue of maytansine. In an embodiment, the cytotoxic or cytostatic agent is a maytansine. Maytansines include structurally complex antimitotic polypeptides. Maytansines are potent inhibitors of microtubulin assembly which leads towards apoptosis of tumour cells. In an embodiment the maytansine is selected from the group consisting of: Mertansine (DM1); and a structural analogue of maytansine such as DM3 or DM4. Preferably, the drug is MMAE, MMAF or auristatin MMAF. In an embodiment the cytotoxic or cytostatic agent is an anti-neoplastic agent such as irinotecan or metabolites thereof. Suitable metabolites of irinotecan include SN-38.

[0316] In an embodiment, the cytotoxic or cytostatic agent is DNA interacting agent. In an embodiment, the DNA interacting agent is selected from the group consisting of: (a) calicheamicins, (b) duocarmycins and (c) pyrrolobenzodiazepines (PBDs). In an embodiment, the cytotoxic or cytostatic agent is a calicheamicin. Calicheamicin is a potent cytotoxic agent that causes doublestrand DNA breaks, resulting in cell death. Calicheamicin is a naturally occurring enediyne antibiotic (A. L. Smith et al, J. Med. Chem., 1996, 39,11 , 2103-21 17). Calicheamicin was found in the soil microorganism Micromonosporaechinospora. In an embodiment, the calicheamicin is calicheamicin gamma 1 . In an embodiment, the drug is a duocarmycin. Duocarmycins are potent anti-tumour antibiotics that exert their biological effects through binding sequence-selectively in the minor groove of DNA duplex and alkylating the N3 of adenine (D. Boger, Pure & Appl. Chem., 1994, 66, 4, 837-844). In an embodiment, the duocarmycin is selected from the group consisting of: Duocarmycin A; Duocarmycin B1 ; Duocarmycin B2; Duocarmycin C1 ; Duocarmycin C2; Duocarmycin D; Duocarmycin SA; Cyclopropylbenzoindole (CBI) duocarmycin; Centanamycin; Rachelmycin (CC-1065); Adozelesin; Bizelesin; and Carzelesin. In an embodiment, the cytotoxic or cytostatic agent is a pyrrolobenzodiazepine. Pyrrolobenzodiazepines (PBDs) are a class of naturally occurring anti-tumour antibiotics. Pyrrolobenzodiazepines are found in Streptomyces. PBDs exert their anti-tumour activity by covalently binding to the DNA in the minor groove specifically at purine-guanine-purine units. They insert on to the N2 of guanine via an aminal linkage and, due to their shape, they cause minimal disruption to the DNA helix. It is believed that the formation of the DNA-PBD adduct inhibits nucleic acid synthesis and causes excisiondependent single and double stranded breaks in the DNA helix. As synthetic derivatives the joining of two PBD units together via a flexible polymethylene tether allows the PBD dimers to cross-link opposing DNA strands producing highly lethal lesions. In an embodiment, the cytotoxic or cytostatic agent is a synthetic derivative of two pyrrolobenzodiazepines units joined together via a flexible polymethylene tether. In an embodiment, the pyrrolobenzodiazepine is selected from the group consisting of: Anthramycin (and dimers thereof); Mazethramycin (and dimers thereof); Tomaymycin (and dimers thereof); Prothracarcin (and dimers thereof); Chicamycin (and dimers thereof); Neothramycin A (and dimers thereof); Neothramycin B (and dimers thereof); DC-81 (and dimers thereof); Sibiromycin (and dimers thereof); Porothramycin A (and dimers thereof); Porothramycin B (and dimers thereof); Sibanomycin (and dimers thereof); Abbeymycin (and dimers thereof); SG3199; SG2000; and SG2285.

[0317] In an embodiment, the cytotoxic or cytostatic agent is a drug that targets DNA interstrand crosslinks through alkylation. A drug that targets DNA interstrand crosslinks through alkylation is selected from: a DNA targeted mustard; a guanine-specific alkylating agent; and a adeninespecific alkylating agent. In an embodiment, the cytotoxic or cytostatic agent is a DNA targeted mustard. For example, the DNA targeted mustard may be selected from the group consisting of: an oligopyrrole; an oligoimidazole; a Bis-(benzimidazole) carrier; a Polybenzamide Carrier; and a 9-Anilinoacridine-4-carboxamide carrier.

[0318] In an embodiment, the cytotoxic or cytostatic agent is selected from the group consisting of: Netropsin; Distamycin; Lexitropsin; Tallimustine; Dibromotallimustine; PNU 157977; and MEN 10710.

[0319] In an embodiment, the cytotoxic or cytostatic agent is a Bis-(benzimidazole) carrier. Preferably, the drug is Hoechst 33258. A guanine-specific alkylating agent is a highly regiospecific alkylating agents that reacts at specific nucleoside positions. In an embodiment, the cytotoxic or cytostatic agent is a guaninespecific alkylating agent selected from the group consisting of: a G-N2 alkylators; a A-N3 alkylator; a mitomycin; a carmethizole analogue; a ecteinascidin analogue. In an embodiment, the mitomycin is selected from: Mitomycin A; Mitomycin C; Porfiromycin; and KW-2149. In an embodiment, the a carmethizole analogue is selected from: Bis-(Hydroxymethyl)pyrrolizidine; and NSC 602668. In an embodiment, the ecteinascidin analogue is Ecteinascidin 743.

[0320] Adenine-specific alkylating agents are regiospecific and sequence-specific minor groove alkylators reacting at the N3 of adenines in polypyrimidines sequences.

[0321] Cyclopropaindolones and duocamycins may be defined as adenine-specific alkylators. In an embodiment, the cytotoxic or cytostatic agent is a cyclopropaindolone analogue. Preferably, the drug is selected from: adozelesin; and carzelesin.

[0322] In an embodiment, the cytotoxic or cytostatic agent is a benz[e]indolone. Preferably, the cytotoxic or cytostatic agent is selected from: CBI-TMI; and iso-CBI.

[0323] In an embodiment, the cytotoxic or cytostatic agent is bizelesin. In an embodiment, the cytotoxic or cytostatic agent is a Marine Antitumour Drug. Marine Antitumour Drugs has been a developing field in the antitumour drug development arena (I. Bhatnagaret al, Mar. Drugs 2010, 8, P2702- 2720 and T. L. Simmons et al, Mol. Cancer Ther. 2005, 4(2), P333-342). Marine organisms including sponges, sponge-microbe symbiotic association, gorgonian, actinomycetes, and soft coral have been widely explored for potential anticancer agents.

[0324] In an embodiment, the cytotoxic or cytostatic agent is selected from: Cytarabine, Ara-C; Trabectedin (ET-743); and EribulinMesylate. In an embodiment, the EribulinMesylate is selected from: (E7389); Soblidotin (TZT 1027); Squalamine lactate; CemadotinPlinabulin (NPI-2358); Plitidepsin; Elisidepsin; Zalypsis; Tasidotin, Synthadotin; (ILX-651); Discodermolide; HT1286; LAF389; Kahalalide F; KRN7000; Bryostatin 1 ; Hemiasterlin (E7974); Marizomib; Salinosporamide A; NPI-0052); LY355703; CRYPTO 52; Depsipeptide (NSC630176); Ecteinascidin 743; Synthadotin; Kahalalide F; Squalamine; Dehydrodidemnin B; Didemnin B; Cemadotin; Soblidotin; E7389; NVP-LAQ824; Discodermolide; HTI-286; LAF-389; KRN-7000 (Agelasphin derivative); Curacin A; DMMC; Salinosporamide A; Laulimalide; Vitilevuamide; Diazonamide; Eleutherobin; Sarcodictyin; Peloruside A; Salicylihalimides A and B; Thiocoraline; Ascididemin; Variolins; Lamellarin D; Dictyodendrins; ES-285 (Spisulosine); and Halichondrin B.

[0325] The following cytotoxic or cytostatic agent are also encompassed by the present invention: Amatoxins (a-amanitin)- bicyclic octapeptides produced by basidiomycetes of the genus Amanita, e.g., the Green Deathcap mushroom; Tubulysins; Pseudomonas exotoxin; Cytolysins; dolabellanins; Epothilone A, B, C, D, E, F. Epothilones - constitute a class of non-taxane tubulin polymerisation agents and are obtained by natural fermentation of the myxobacterium Sorangiumcellulosum. These moieties possess potent cytotoxic activity which is linked to the stabilisation of microtubules and results in mitotic arrest at the G2 / M transition. Epothilones have demonstrated potent cytotoxicity across a panel of cancer cell lines and has often exhibited greater potency than paclitaxel (X. :Pivot et al, European Oncology, 2008;4(2), P42-45). Pseudomonas exotoxin is an exotoxin produced by Pseudomonas aeruginosa which catalyzes the ADP-ribosylation and inactivation of EF2, which leads to protein synthesis inhibition and cell death. In an embodiment, the drug or payload is amatoxin. In an embodiment, the drug or payload is tubulysin. In an embodiment the drug or payload is Pseudomonas exotoxin. In an embodiment, the drug or payload is cytolysin. In an embodiment, the drug or payload is dolabellanin. In an embodiment, the drug or payload is epothilone.

[0326] The following cytotoxic or cytostatic agent are also encompassed by the present invention. In an embodiment, the drug is selected from: Doxorubicin; Epirubicin; Esorubicin; Detorubicin; Morpholino-doxorubicin; Methotrexate; Methopterin; Bleomycin; Dichloromethotrexate; 5- Fluorouracil; Cytosine-p-D-arabinofuranoside; Taxol; Anguidine; Melphalan; Vinblastine; Phomopsin A; Ribosome-inactivating proteins (RIPs); Daunorubicin; Vinca alkaloids; Idarubicin; Melphalan; Cis-platin; Ricin; Saporin; Anthracyclines; Indolino-benzodiazepines; 6- Mercaptopurine; Actinomycin; Leurosine; Leurosideine; Carminomycin; Aminopterin; Tallysomycin; Podophyllotoxin; Etoposide; Hairpin polyamides; Etoposide phosphate; Vinblastine; Vincristine; Vindesine; Taxotere retinoic acid; N8-acetyl spermidine; Camptothecin; Esperamicin; and Ene-diynes.

[0327] In one embodiment, the cell killing portion is a peptide toxin, for example an auristatin such as MMAE or MMAF. In one embodiment, the bispecific binding molecule comprises a binding portion and a cell killing portion, wherein the binding portion is an anti-CD33 anti-CD7 bispecific antibody or binding portion thereof and wherein the cell killing portion is a peptide toxin, for example an auristatin such as Auristatin E (AE); Monomethylauristatin E (MMAE); Auristatin F (MMAF), vcMMAE, vcMMAF, mcMMAE and mcMMAF.

[0328] In certain embodiments, the antibody or antigen binding fragments thereof, comprises a binding portion that is conjugated to a payload, for example a cell killing agent, an immune-modulating payload, a macrophage class switching agent or a light activatable payload. Such conjugates may be prepared by in vitro methods known to one of ordinary skill in the art. Techniques for conjugating cytotoxic or cytostatic agent to proteins, and in particular to antibodies, are well- known. (See, e.g., Alley et ah, Current Opinion in Chemical Biology 2010 14: 1-9; Senter, Cancer J., 2008, 14(3): 154-169.) In certain embodiments the payload comprises a small molecule inhibitor with an anti-cancer activity. For example the small molecule inhibitor may be a Bcl-XI inhibitor, Bcl2 inhibitor, Bcl-w inhibitor, Bcr-Abl inhibitor, EGFR inhibitor VEGFR2 inhibitor, RET inhibitor, PDGFR inhibitor, FLT-3 inhibitor, KIT inhibitor, CSF-1 inhibitor, HER2 inhibitor, LCK inhibitor, B-raf inhibitor, mTOR inhibitor, c-KIT inhibitor, FGFR inhibitor, VEGFR inhibitor, HGFR inhibitor, Jak1 inhibitor, Jak2 inhibitor, VEGFR1-3 inhibitor, Src inhibitor, c.MET inhibitor, PDGFR-p inhibitor, MEK inhibitor, HSP90 inhibitor, MMP inhibitor, proteosome inhibitor, Akt inhibitor, NAMPT inhibitor.

[0329] In certain embodiments the payload comprises a light-activatable payload, for example a infrared light activatable payload. Immunoconjugates comprising a near-infrared activatable payload enable binding molecule-mediated targeted delivery to achieve a high degree of tumour specificity, while using infrared light to activate the biophysical mechanism of the drug to accurately induce rapid death of cancer cells without harming the surrounding normal tissues. Suitable light-activatable payloads include IRDye700DX. In one embodiment a light activatable payload (IRDye® 700DX, IR700) may also be used. Light activation of the non-toxic payload results in the generation of singlet oxygen species that damage the cell membrane integrity, resulting in necrotic and immunogenic cell death of tumour cells, resulting in minimal damage to surrounding normal tissue.

[0330] Wherein the bispecific binding molecule is provided conjugated to a payload the first antigen binding domain may have a binding affinity (KD) to CD7 between 5 x10-10M and 2 x10-7M, between 5 x10-9M and 2 x10-7M, between 5 x10-1° M and 2 x10-8M, between 5 x10-9M and 2 x10-9M, of between 6.5 x10-1° M and 2 x10-7M, between 6.5 x10-9M and 2 x10-7M, between 6.5 x10wM and 2 x108M, between 6.5 x10wM and 2 x109M, between 6.5 x109M and 2 x10’8M.

[0331] The bispecific binding molecule or the immunoconjugate of the invention may comprise an IC50 in the range of 0.005 to 5 nM, 0.006 to 5 nM, 0.007 to 5 nM, 0.008 to 5 nM, 0.009 to 5 nM, 0.01 to 5 nM, 0.005 to 4.5 nM, 0.006 to 4.5 nM, 0.007 to 4.5 nM, 0.008 to 4.5 nM, 0.009 to 4.5 nM, 0.01 to 4.5 nM, 0.005 to 4 nM, 0.006 to 4 nM, 0.007 to 4 nM, 0.008 to 4 nM, 0.009 to 4 nM, 0.01 to 4 nM, 0.005 to 3.5 nM, 0.006 to 3.5 nM, 0.007 to 3.5 nM, 0.008 to 3.5 nM, 0.009 to 3.5 nM, 0.01 to 3.5 nM, 0.005 to 3 nM, 0.006 to 3 nM, 0.007 to 3 nM, 0.008 to 3 nM, 0.009 to 3 nM, 0.01 to 3 nM, 0.005 to 2.5 nM, 0.006 to 2.5 nM, 0.007 to 2.5 nM, 0.008 to 2.5 nM, 0.009 to 2.5 nM, 0.01 to 2.5 nM, 0.005 to 2 nM, 0.006 to 2 nM, 0.007 to 2 nM, 0.008 to 2 nM, 0.009 to 2 nM, 0.01 to 2 nM, 0.005 to 1.5 nM, 0.006 to 1.5 nM, 0.007 to 1.5 nM, 0.008 to 1.5 nM, 0.009 to 1.5 nM, 0.01 to 1 .5 nM, or 0.02 to 1 .2 nM The bispecific binding molecule or the immunoconjugate of the invention may comprise an IC50 for dual expressing cells i.e. CD7+CD33+ in the range of 0.005 to 5 nM, 0.006 to 5 nM, 0.007 to 5 nM, 0.008 to 5 nM, 0.009 to 5 nM, 0.01 to 5 nM, 0.005 to 4.5 nM, 0.006 to 4.5 nM, 0.007 to 4.5 nM, 0.008 to 4.5 nM, 0.009 to 4.5 nM, 0.01 to 4.5 nM, 0.005 to 4 nM, 0.006 to 4 nM, 0.007 to 4 nM, 0.008 to 4 nM, 0.009 to 4 nM, 0.01 to 4 nM, 0.005 to 3.5 nM, 0.006 to 3.5 nM, 0.007 to 3.5 nM, 0.008 to 3.5 nM, 0.009 to 3.5 nM, 0.01 to 3.5 nM, 0.005 to 3 nM, 0.006 to 3 nM, 0.007 to 3 nM, 0.008 to 3 nM, 0.009 to 3 nM, 0.01 to 3 nM, 0.005 to 2.5 nM, 0.006 to 2.5 nM, 0.007 to 2.5 nM, 0.008 to 2.5 nM, 0.009 to 2.5 nM, 0.01 to 2.5 nM, 0.005 to 2 nM, 0.006 to 2 nM, 0.007 to 2 nM, 0.008 to 2 nM, 0.009 to 2 nM, 0.01 to 2 nM, 0.005 to 1 .5 nM, 0.006 to 1 .5 nM, 0.007 to 1 .5 nM, 0.008 to 1 .5 nM, 0.009 to 1 .5 nM, 0.01 to 1 .5 nM, or 0.02 to 1 .2 nM

[0332] Techniques for conjugating cytotoxic or cytostatic agents to proteins, and in particular to antibodies, are well-known. (See, e.g., Alley et ah, Current Opinion in Chemical Biology 2010 14: 1-9; Senter, Cancer J., 2008, 14(3): 154-169.). In certain embodiments, a linking group is used to conjugate the payload, for example a cell killing agent, an immune-modulating payload, a macrophage class switching agent or a light activatable payload, to the first and / or second antibody or antigen-binding fragment, as appropriate. The linker can be cleavable under intracellular conditions, such that cleavage of the linker releases the payload from the binding portion in the intracellular environment. The cleavable linker can be, e.g., a peptidyl linker that is cleaved by an intracellular peptidase or protease enzyme, including a lysosomal or endosomal protease. Cleaving agents can include cathepsins B and D and plasmin (see, e.g., Dubowchik and Walker, Pharm. Therapeutics 83:67-123, 1999). Most typical are peptidyl linkers that are cleavable by enzymes that are present in NTB-A-expressing cells. For example, a peptidyl linker that is cleavable by the thiol-dependent protease cathepsin-B, which is highly expressed in cancerous tissue, can be used (e.g., a linker comprising a Phe-Leu or a Val-Cit peptide).

[0333] In certain embodiments, a linking group is used to conjugate the binding portion and the payload, for example a cell killing agent, an immune-modulating payload, a macrophage class switching agent or a light activatable payload.

[0334] The payload can be linked to an antibody by a linker. Attachment of a linker to a mAb can be accomplished in a variety of ways, such as through surface lysines, reductive- coupling to oxidized carbohydrates, and through cysteine residues liberated by reducing interchain disulfide linkages. A variety of linkage systems are known in the art, including hydrazone-, disulfide- and peptide-based linkages.

[0335] Suitable linkers include, for example, cleavable and non-cleavable linkers. A cleavable linker is typically susceptible to cleavage under intracellular conditions. Suitable cleavable linkers include, for example, a peptide linker cleavable by an intracellular protease, such as lysosomal protease or an endosomal protease. In exemplary embodiments, the linker can be a dipeptide linker, such as a valine-citrulline (val-cit), a phenylalanine-lysine (phe-lys) linker, or maleimidocapronic-valine-citruline-p-aminobenzyloxycarbonyl (mc-Val-Cit- PABA) linker. Another linker is Sulfosuccinimidyl-4-[N-maleimidomethyl]cyclohexane-l- carboxylate (smcc). Sulfo-smcc conjugation occurs via a maleimide group which reacts with sulfhydryls (thiols, — SH), while its Sulfo-NHS ester is reactive toward primary amines (as found in Lysine and the protein or peptide N-terminus). Yet another linker is maleimidocaproyl (me). Other suitable linkers include linkers hydrolyzable at a specific pH or a pH range, such as a hydrazone linker. Additional suitable cleavable linkers include disulfide linkers. The linker may be covalently bound to the antibody to such an extent that the antibody must be degraded intracellularly in order for the drug to be released e.g. the me linker and the like.

[0336] Thus, a linker can include a group for linkage to the antibody. For example, linker can include an amino, hydroxyl, carboxyl or sulfhydryl reactive groups (e.g., malemide, haloacetamides (e.g., iodo, bromo or chloro), haloesters (e.g., iodo, bromo or chloro), halomethyl ketones (e.g., iodo, bromo or chloro), benzylic halides (e.g., iodide, bromide or chloride), vinyl sulfone and pyridylthio). See generally Wong, Chemistry of Protein

[0337] The linker can be cleavable under intracellular conditions, such that cleavage of the linker releases the payload from the binding portion in the intracellular environment. The cleavable linker can be, e.g., a peptidyl linker that is cleaved by an intracellular peptidase or protease enzyme, including a lysosomal or endosomal protease. Cleaving agents can include cathepsins B and D and plasmin (see, e.g., Dubowchik and Walker, Pharm. Therapeutics 83:67-123, 1999). Most typical are peptidyl linkers that are cleavable by enzymes that are present in NTB-A- expressing cells. For example, a peptidyl linker that is cleavable by the thiol-dependent protease cathepsin-B, which is highly expressed in cancerous tissue, can be used (e.g., a linker comprising a Phe-Leu or a Val-Cit peptide).

[0338] The cleavable linker can be pH-sensitive, i.e., sensitive to hydrolysis at certain pH values. Typically, the pH- sensitive linker is hydrolysable under acidic conditions. For example, an acid- labile linker that is hydrolysable in the lysosome (e.g., a hydrazone, semicarbazone, thiosemicarbazone, cis-aconitic amide, orthoester, acetal, ketal, or the like) can be used.

[0339] Other linkers are cleavable under reducing conditions (e.g., a disulfide linker). The cleavable linker can also be a malonate linker (Johnson et al, Anticancer Res. 15 :1387-93, 1995), a maleimidobenzoyl linker (Lau et al, Bioorg-Med-Chem. 3: 1299-1304, 1995), or a 3' -N-amide analogue (Lau et al, Bioorg-Med-Chem. 3: 1305-12, 1995). In some embodiments the linker can be a protease cleavable linker, for example a valinecitrulline, which may be cleaved by cathepsin B in the lysosome.

[0340] The linker also can be a non-cleavable linker, such as a maleimidoca-proyl (me) linker or maleimido-alkylene- or maleimide-aryl linker that is directly attached to the therapeutic agent and released by proteolytic degradation of the binding portion.

[0341] The terms “conjugation” and “conjugate(d)” refer to chemical linkages, either covalent or non- covalent, which proximally associates one molecule of interest with a second molecule of interest. The drug-to- antibody ratio (DAR) can be, e.g., 1 .0 to 8.0, 1 to 7.0, 1 .0 to 6.0, 1 .0 to 5.0, 1.0 to 4.0, 1.0 to 3.0. 1 .0 to 2.0.

[0342] The conjugate may be prepared by several routes, employing organic chemistry reactions, conditions, and reagents known to those skilled in the art, including: (1) reaction of a nucleophilic group or an electrophilic group of an antibody with a bivalent linker reagent, to form antibodylinker intermediate Ab-L, via a covalent bond, followed by reaction with an activated drug moiety D; and (2) reaction of a nucleophilic group or an electrophilic group of a drug moiety with a linker reagent, to form drug-linker intermediate D-L, via a covalent bond, followed by reaction with the nucleophilic group or an electrophilic group of an antibody. Conjugation methods (1) and (2) may be employed with a variety of antibodies, drug moieties, and linkers to prepare the antibodydrug conjugates described here.

[0343] The bispecific binding molecule may comprise a first and second antigen binding domain wherein the first antigen binding domain comprises i) a heavy chain variable (VH) region comprising SEQ ID NO. 21 or a sequence comprising at least 90%, 92%, 95%, 97% or 99% sequence identity thereto, and ii) a light chain variable (VL) region comprising SEQ ID NO. 23 or a sequence comprising at least 80%, 85%, 90%, 92%, 95%, 97% or 99% sequence identity thereto, and the second antigen binding domain comprises i) a heavy chain variable (VH) region comprising SEQ ID NO. 1 and ii) a light chain variable (VL) region comprising SEQ ID NO. 6 wherein the bispecific binding molecule is conjugated to one or more of the payloads described herein.

[0344] The bispecific molecules of the present invention may be formed by any suitable method, for example a bispecific antibody may be recombinantly expressed using standard techniques known in the art, or a bispecific molecule may be formed by linking the first and second antigen binding domain together. Methods of producing bispecific molecule are known in the art. Several specific examples of methods of preparing bispecific antibodies ADCs are known in the art. Traditional methods such as the hybrid hybridoma and chemical conjugation methods can be used in the preparation of the bispecific antibodies of the invention. Co-expression in a host cell of two antibodies, consisting of different heavy and light chains, leads to a mixture of possible antibody products in addition to the desired bispecific antibody, which can then be isolated by, e.g., affinity chromatography or similar methods.

[0345] Strategies favoring the formation of a functional bispecific, product, upon co-expression of different antibody constructs can also be used. Strategies for promoting heterodimerization are known in the art. One strategy to promote formation of heterodimers over homodimers is a "knob-into-hole" strategy in which a protuberance is introduced on a first heavy-chain polypeptide and a corresponding cavity in a second heavy-chain polypeptide, such that the protuberance can be positioned in the cavity at the interface of these two heavy chains so as to promote heterodimer formation and hinder homodimer formation.

[0346] Nucleophilic groups on antibodies include, but are not limited to: (i) N-terminal amine groups, (ii) side chain amine groups, e.g. lysine, (iii) side chain thiol groups, e.g. cysteine, and (iv) sugar hydroxyl or amino groups where the antibody is glycosylated. Amine, thiol, and hydroxyl groups are nucleophilic and capable of reacting to form covalent bonds with electrophilic groups on linker moieties and linker reagents including: (i) active esters such as NHS esters, HOBt esters, haloformates, and acid halides; (ii) alkyl and benzyl halides such as haloacetamides; (iii) aldehydes, ketones, carboxyl, and maleimide groups. Certain antibodies have reducible interchain disulfides, i.e. cysteine bridges. Antibodies may be made reactive for conjugation with linker reagents by treatment with a reducing agent.

[0347] Additional nucleophilic groups can be introduced into antibodies through the reaction of lysines with 2-iminothiolane (Traut's reagent) resulting in conversion of an amine into a thiol.

[0348] Antibody-drug conjugates may also be produced by modification of the antibody to introduce electrophilic moieties, which can react with nucleophilic substituents on the linker reagent or drug. The sugars of glycosylated antibodies may be oxidized, e.g. with periodate oxidizing reagents, to form aldehyde or ketone groups which may react with the amine group of linker reagents or drug moieties. The resulting imine Schiff base groups may form a stable linkage, or may be reduced, e.g. by borohydride reagents to form stable amine linkages. In one embodiment, reaction of the carbohydrate portion of a glycosylated antibody with either galactose oxidase or sodium meta-periodate may yield carbonyl (aldehyde and ketone) groups in the protein that can react with appropriate groups on the drug. In another embodiment, proteins containing N-terminal serine or threonine residues can react with sodium meta- periodate, resulting in production of an aldehyde in place of the first amino acid. Such aldehyde can be reacted with a drug moiety or linker nucleophile.

[0349] Likewise, nucleophilic groups on a drug moiety include, but are not limited to: amine, thiol, hydroxyl, hydrazide, oxime, hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide groups capable of reacting to form covalent bonds with electrophilic groups on linker moieties and linker reagents including: (i) active esters such as NHS esters, HOBt esters, haloformates, and acid halides; (ii) alkyl and benzyl halides such as haloacetamides; (iii) aldehydes, ketones, carboxyl, and maleimide groups.

[0350] Methods for conjugating the payload to the bispecific binding molecule may utilise the presence of one or more non-naturally occurring amino acids i.e. a non-wild-type residue. The payload and / or linker may be attached to the bispecific binding molecule via a non-naturally occurring amino acid residue present in the bispecific antibody or antigen binding fragment. Methods to introduce non-naturally occurring amino acid residues are known in the art.

[0351] The non-naturally occurring amino acid residue to which the payload and / or linker is attached is may be selected from but not limited to a cysteine residue, a lysine residue, a histidine residue, a tyrosine residue, formylglycine residue.

[0352] Where a canonical amino acid is used to attach the payload and / or linker, site directed mutagenesis may be used to introduce a suitable amino acid residue at a suitable position within the binding molecule. In an embodiment a non-canonical amino acid is used to attach the payload linker. A “non-canonical” amino acid refers to one of the non-proteinogenic (unnatural) amino acids i.e., an amino acid which is not introduced via the cell’s natural translation machinery. There are many examples of non-canonical amino acids in the art many of which provide a bio-orthogonal handle on which to attach a payload. Where a non-canonical amino acid is used to attach the payload linker, suitable techniques are known in the art to introduce such non-canonical amino acid residues such as chemical modification, tRNA suppressor technology, engineered tRNA, / tRNA synthetase pairs.

[0353] The payload may be attached at various positions within the bispecific molecule. One or more copies of the payload may be attached to the molecule, for example 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2 copies of the payload may be attached.

[0354] The position at which the payload is attached to the bispecific binding molecule may directed by the position at which the non-naturally occurring amino acid is introduced or the position at which the electrophilic moiety is introduced. As such site-specific attachments of the payload can be achieved. In another embodiment, the second moity is a label, for example a fluorescent molecule, p- galactosidase, luciferase molecules, chemical dyes, fluorophores or a radioisotope.

[0355] Where the binding molecules of the invention are provided as an immunoconjugate or an ADC they demonstrates low payload deconjugation e.g. <10%. The immunoconjugate or ADC may comprise a payload deconjugation of <15%, <14%, <13%, <12%, <1 1 %, <10%, <9%, <8%, <7%, <6%, <5%, <4%, <3%, <2%, when assessed over 5 days.

[0356] In an embodiment the bispecific binding molecule, comprises a first antigen binding domain that binds to CD7 and a second antigen binding domain that binds CD33, wherein said first antigen binding domain comprises i) a heavy chain variable (VH) region comprising SEQ ID NO. 21 or a sequence comprising at least 90%, 92%, 95%, 97% or 99% sequence identity thereto, and ii) a light chain variable (VL) region comprising SEQ ID NO. 23 or a sequence comprising at least 80%, 85%, 90%, 92%, 95%, 97% or 99% sequence identity thereto, wherein the bispecific molecule comprises one or more of the following additional features a) the bispecific binding molecule is capable of targeting cells expressing CD7+CD33+, wherein said cells express CD33+ at a low level, b) a second antigen binding domain comprising i) a heavy chain variable (VH) region comprising a HC CDR1 comprising SEQ ID NO. 2, a HC CDR2 comprising SEQ ID NO. 3, a HC CDR3 comprising SEQ ID NO. 4, and ii) a light chain variable (VL) region comprising a LC CDR1 comprising SEQ ID NO. 7, a LC CDR2 comprising SEQ ID NO. 8, a LC CDR3 comprising SEQ ID NO. 9, c) the first and / or second antigen binding domain is a Fab domain, d) the first and / or second antigen binding domain comprises a thiol containing group at one of more of positions 153, 156, 168, 173 and / or 207 according to Kabat numbering, e) the first and second antigen binding domain are conjugated together via clickchemistry, f) the first and second antigen binding domains are conjugated together via a linker which attaches the first and second antigen-binding domain, wherein the linker is attached to each of the first and second sulphur atoms, S’ and S”, of the first and second antigen-binding domain, respectively, and / or g) the bispecific binding molecule is conjugated to one or more of the payloads described herein.

[0357] In an embodiment the bispecific binding molecule, comprises a first antigen binding domain that binds to CD7 and a second antigen binding domain that binds CD33, wherein said first antigen binding domain comprises i) a heavy chain variable (VH) region comprising SEQ ID NO. 25 or a sequence comprising at least 90%, 92%, 95%, 97% or 99% sequence identity thereto, and ii) a light chain variable (VL) region comprising SEQ ID NO. 27 or a sequence comprising at least 80%, 85%, 90%, 92%, 95%, 97% or 99% sequence identity thereto, wherein the bispecific molecule comprises one or more of the following additional features a) the bispecific binding molecule is capable of targeting cells expressing CD7+CD33+, wherein said cells express CD33+ at a low level, b) a second antigen binding domain comprising i) a heavy chain variable (VH) region comprising a HC CDR1 comprising SEQ ID NO. 2, a HC CDR2 comprising SEQ ID NO. 3, a HC CDR3 comprising SEQ ID NO. 4, and ii) a light chain variable (VL) region comprising a LC CDR1 comprising SEQ ID NO. 7, a LC CDR2 comprising SEQ ID NO. 8, a LC CDR3 comprising SEQ ID NO. 9, c) the first and / or second antigen binding domain is a Fab domain, d) the first and / or second antigen binding domain comprises a thiol containing group at one of more of positions 153, 156, 168, 173 and / or 207 according to Kabat numbering, e) the first and second antigen binding domain are conjugated together via clickchemistry, f) the first and second antigen binding domains are conjugated together via a linker which attaches the first and second antigen-binding domain, wherein the linker is attached to each of the first and second sulphur atoms, S’ and S”, of the first and second antigen-binding domain, respectively, and / or g) the bispecific binding molecule is conjugated to one or more of the payloads described herein.

[0358] In an embodiment the bispecific binding molecule, comprises a first antigen binding domain that binds to CD7 and a second antigen binding domain that binds CD33, wherein said first antigen binding domain comprises i) a heavy chain variable (VH) region comprising SEQ ID NO. 29 or a sequence comprising at least 90%, 92%, 95%, 97% or 99% sequence identity thereto, and ii) a light chain variable (VL) region comprising SEQ ID NO. 31 or a sequence comprising at least 80%, 85%, 90%, 92%, 95%, 97% or 99% sequence identity thereto, wherein the bispecific molecule comprises one or more of the following additional features a) the bispecific binding molecule is capable of targeting cells expressing CD7+CD33+, wherein said cells express CD33+ at a low level, b) a second antigen binding domain comprising i) a heavy chain variable (VH) region comprising a HC CDR1 comprising SEQ ID NO. 2, a HC CDR2 comprising SEQ ID NO. 3, a HC CDR3 comprising SEQ ID NO. 4, and ii) a light chain variable (VL) region comprising a LC CDR1 comprising SEQ ID NO. 7, a LC CDR2 comprising SEQ ID NO. 8, a LC CDR3 comprising SEQ ID NO. 9, c) the first and / or second antigen binding domain is a Fab domain, d) the first and / or second antigen binding domain comprises a thiol containing group at one of more of positions 153, 156, 168, 173 and / or 207 according to Kabat numbering, e) the first and second antigen binding domain are conjugated together via clickchemistry, f) the first and second antigen binding domains are conjugated together via a linker which attaches the first and second antigen-binding domain, wherein the linker is attached to each of the first and second sulphur atoms, S’ and S”, of the first and second antigen-binding domain, respectively, and / or g) the bispecific binding molecule is conjugated to one or more of the payloads described herein.

[0359] In an embodiment the bispecific binding molecule, comprises a first antigen binding domain that binds to CD7 and a second antigen binding domain that binds CD33, wherein said first antigen binding domain comprises i) a heavy chain variable (VH) region comprising SEQ ID NO. 33 or a sequence comprising at least 90%, 92%, 95%, 97% or 99% sequence identity thereto, and ii) a light chain variable (VL) region comprising SEQ ID NO. 35 or a sequence comprising at least 80%, 85%, 90%, 92%, 95%, 97% or 99% sequence identity thereto, wherein the bispecific molecule comprises one or more of the following additional features a) the bispecific binding molecule is capable of targeting cells expressing CD7+CD33+, wherein said cells express CD33+ at a low level, b) a second antigen binding domain comprising i) a heavy chain variable (VH) region comprising a HC CDR1 comprising SEQ ID NO. 2, a HC CDR2 comprising SEQ ID NO. 3, a HC CDR3 comprising SEQ ID NO. 4, and ii) a light chain variable (VL) region comprising a LC CDR1 comprising SEQ ID NO. 7, a LC CDR2 comprising SEQ ID NO. 8, a LC CDR3 comprising SEQ ID NO. 9, c) the first and / or second antigen binding domain is a Fab domain, d) the first and / or second antigen binding domain comprises a thiol containing group at one of more of positions 153, 156, 168, 173 and / or 207 according to Kabat numbering, e) the first and second antigen binding domain are conjugated together via clickchemistry, f) the first and second antigen binding domains are conjugated together via a linker which attaches the first and second antigen-binding domain, wherein the linker is attached to each of the first and second sulphur atoms, S’ and S”, of the first and second antigen-binding domain, respectively, and / or g) the bispecific binding molecule is conjugated to one or more of the payloads described herein.

[0360] In an embodiment the bispecific binding molecule, comprises a first antigen binding domain that binds to CD7 and a second antigen binding domain that binds CD33, wherein said first antigen binding domain comprises i) a heavy chain variable (VH) region comprising SEQ ID NO. 37 or a sequence comprising at least 90%, 92%, 95%, 97% or 99% sequence identity thereto, and ii) a light chain variable (VL) region comprising SEQ ID NO. 39 or a sequence comprising at least 80%, 85%, 90%, 92%, 95%, 97% or 99% sequence identity thereto wherein the bispecific molecule comprises one or more of the following additional features a) the bispecific binding molecule is capable of targeting cells expressing CD7+CD33+, wherein said cells express CD33+ at a low level, b) a second antigen binding domain comprising i) a heavy chain variable (VH) region comprising a HC CDR1 comprising SEQ ID NO. 2, a HC CDR2 comprising SEQ ID NO. 3, a HC CDR3 comprising SEQ ID NO. 4, and ii) a light chain variable (VL) region comprising a LC CDR1 comprising SEQ ID NO. 7, a LC CDR2 comprising SEQ ID NO. 8, a LC CDR3 comprising SEQ ID NO. 9, c) the first and / or second antigen binding domain is a Fab domain, d) the first and / or second antigen binding domain comprises a thiol containing group at one of more of positions 153, 156, 168, 173 and / or 207 according to Kabat numbering, e) the first and second antigen binding domain are conjugated together via clickchemistry, f) the first and second antigen binding domains are conjugated together via a linker which attaches the first and second antigen-binding domain, wherein the linker is attached to each of the first and second sulphur atoms, S’ and S”, of the first and second antigen-binding domain, respectively, and / or g) the bispecific binding molecule is conjugated to one or more of the payloads described herein.

[0361] Nucleic Acid, Vectors, Host Cells

[0362] An aspect of the invention relates to an isolated nucleic acid molecule encoding the bispecific binding molecule disclosed herein. The isolated nucleic acid may be encoded in a construct for example a plasmid, vector, transcription or expression cassette. In an embodiment the nucleic acid is provided in a vector. Any suitable vector may be used.

[0363] The isolated nucleic acid or the construct comprising said nucleic acid may be provided in a host cell. The host cell may be a bacterial, viral, plant, mammalian or another suitable host cell. The host cell comprising said vector or isolated nucleic acid may be used to produce the bispecific binding molecule. There are several methods by which to produce recombinant antibodies which are known in the art. One of these is production in an E. coli expression system. In this embodiment, nucleic acids encoding the antibody or antigen-binding fragment thereof as described in previous aspects of the invention may be inserted into a plasmid and expressed in a suitable expression system. For example, the present invention includes methods for expressing a binding molecule in a host cell (e.g., bacterial host cell such as E. coli, CHO, HEK or other host cell according to the above described aspects of the invention).

[0364] Such host cells are well known in the art and many are available from the American Type Culture Collection (ATCC). These host cells include, inter alia, Chinese hamster ovary (CHO) cells, such as ExpiCHO cells, NSO, SP2 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, 3T3 cells, HEK- 293 cells and a number of other cell lines. Mammalian host cells include human, mouse, rat, dog, monkey, pig, goat, bovine, horse and hamster cells. Other cell lines that may be used are insect cell lines (e.g., Spodoptera frugiperda or Trichoplusia ni), amphibian cells, bacterial cells, plant cells and fungal cells. Fungal cells include yeast and filamentous fungus cells including, for example, Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia minuta (Ogataea minuta, Pichia lindneri), Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichia methanolica, Pichia sp., Saccharomyces cerevisiae, Saccharomyces sp., Hansenula polymorpha, Kluyveromyces sp., Kluyveromyces lactis, Candida albicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chrysosporium lucknowense, Fusarium sp., Fusarium gramineum, Fusarium venenatum, Physcomitrella patens and Neurospora crassa.

[0365] Transformation can be by any known method for introducing polynucleotides into a host cell. Methods for introduction of heterologous polynucleotides into mammalian cells are well known in the art and include dextran-mediated transfection, calcium phosphate precipitation, polybrene- mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide(s) in liposomes, biolistic injection and direct microinjection of the DNA into nuclei. In addition, nucleic acid molecules may be introduced into mammalian cells by viral vectors. Methods of transforming cells are well known in the art.

[0366] Pharmaceutical Composition

[0367] The bispecific binding molecule may be provided as a pharmaceutical composition. The pharmaceutical composition may optionally comprise a pharmaceutically acceptable excipient, and / or adjuvant. The pharmaceutical compositions described herein may comprise the bispecific binding molecule. The pharmaceutical compositions described herein may comprise the immunoconjugate or the ADC comprising said bispecific binding molecule. The pharmaceutical compositions described herein can be administered by any convenient route, including but not limited to oral, topical, parenteral, sublingual, rectal, vaginal, ocular, intranasal, pulmonary, intradermal, intravitrial, intratumoural, intramuscular, intraperitoneal, intravenous, subcutaneous, intracerebral, transdermal, transmucosal, by inhalation, or topical, particularly to the ears, nose, eyes, or skin or by inhalation. In another embodiment, delivery is of the nucleic acid encoding the drug, e.g. a nucleic acid encoding the molecule of the invention is delivered.

[0368] Parenteral administration includes, for example, intravenous, intramuscular, intraarterial, intraperitoneal, intranasal, rectal, intravesical, intradermal, topical, intra-articular or subcutaneous administration. In an embodiment, the compositions are administered parenterally.

[0369] The pharmaceutically acceptable carrier or vehicle can be particulate, so that the compositions are, for example, in tablet or powder form. The term "carrier" refers to a diluent, adjuvant or excipient, with which a binding molecule of the present invention is administered. Suitable diluents, adjuvants and excipients can 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. The carriers can be saline, gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea, and the like. In addition, auxiliary, stabilizing, thickening, lubricating and coloring agents can be used. In one embodiment, when administered to an animal, the bispecific antibody or antigen binding portion thereof of the present invention or compositions and pharmaceutically acceptable carriers are sterile. Water is a preferred carrier when the drug antibody conjugates of the present invention are administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical carriers also include excipients such as starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The present compositions, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.

[0370] The pharmaceutical composition can be in the form of a liquid, e.g., a solution, syrup, solution, emulsion or suspension. The liquid can be useful for oral administration or for delivery by injection, infusion (e.g., IV infusion) or sub-cutaneous. When intended for oral administration, the composition can be in solid or liquid form, where semisolid, semi-liquid, suspension and gel forms are included within the forms considered herein as either solid or liquid.

[0371] As a solid composition for oral administration, the composition can be formulated into a powder, granule, compressed tablet, pill, capsule, chewing gum, wafer or the like form. Such a solid composition typically contains one or more inert diluents. In addition, one or more of the following can be present: binders such as carboxymethylcellulose, ethyl cellulose, microcrystalline cellulose, or gelatin; excipients such as starch, lactose or dextrins, disintegrating agents such as alginic acid, sodium alginate, corn starch and the like; lubricants such as magnesium stearate; glidants such as colloidal silicon dioxide; sweetening agents such as sucrose or saccharin; a flavoring agent such as peppermint, methyl salicylate or orange flavoring; and a coloring agent. When the composition is in the form of a capsule (e. g. a gelatin capsule), it can contain, in addition to materials of the above type, a liquid carrier such as polyethylene glycol, cyclodextrin or a fatty oil.

[0372] When intended for oral administration, a composition can comprise one or more of a sweetening agent, preservatives, dye / colorant and flavour enhancer. In a composition for administration by injection, one or more of a surfactant, preservative, wetting agent, dispersing agent, suspending agent, buffer, stabilizer and isotonic agent can also be included.

[0373] Compositions can take the form of one or more dosage units.

[0374] In specific embodiments, it can be desirable to administer the composition locally to the area in need of treatment, or by intravenous injection or infusion.

[0375] The amount of the bispecific binding molecule or pharmaceutical composition described herein that is effective / active in the treatment of a particular disease or condition will depend on the nature of the disease or condition and can be determined by standard clinical techniques. In addition, in vitro or in vivo assays can optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the compositions will also depend on the route of administration, and the seriousness of the disease or disease, and should be decided according to the judgment of the practitioner and each patient's circumstances. Factors like age, body weight, sex, diet, time of administration, rate of excretion, condition of the host, drug combinations, reaction sensitivities and severity of the disease shall be taken into account.

[0376] Therapy

[0377] The molecules of the present invention are capable of targeting specific cell populations found within malignant cell populations, as such they have utility in therapy. An aspect of the invention relates to the bispecific binding molecule, immunoconjugate or antibody drug conjugate described herein for use in the treatment of a malignancy.

[0378] An aspect of the invention relates to a method of treating or preventing a malignancy comprising administering a therapeutically effective amount of the bispecific binding molecule, immunoconjugate or antibody drug conjugate described herein.

[0379] An aspect of the invention relates to the use of the bispecific binding molecule, immunoconjugate or antibody drug conjugate described herein for the manufacture of a medicament for the treatment of a malignancy.

[0380] The term malignancy as used herein refers to any disease sate comprising the uncontrolled growth and division of abnormal cells, for example cancer. The binding molecules of the invention are for use in the treatment of disease state comprising malignant cellular proliferation including but not limited to neoplasms and tumours (e.g., histocytoma, glioma, astrocyoma, osteoma), cancers (e.g. lung cancer, small cell lung cancer, gastrointestinal cancer, bowel cancer, colon cancer, breast carinoma, ovarian carcinoma, prostate cancer, testicular cancer, liver cancer, kidney cancer, bladder cancer, pancreatic cancer, brain cancer, sarcoma, osteosarcoma, Kaposi's sarcoma, melanoma), leukemias, psoriasis, bone diseases, fibroproliferative disorders (e.g. of connective tissues), and atherosclerosis. In certain embodiments the binding molecules are for use in haematological; malignancies such as leukemias and lymphomas, such as non-Hodgkin lymphoma, and subtypes such as DLBCL, marginal zone, mantle zone, and follicular, Hodgkin lymphoma, Acute Myeloid Leukaemia (AML) and other cancers of B or T cell origin. Any type of cell may be treated, including but not limited to, lung, gastrointestinal (including, e.g. bowel, colon), breast (mammary), ovarian, prostate, liver (hepatic), kidney (renal), bladder, pancreas, brain, and skin.

[0381] The malignancy may be a haematological malignancy. Haematologic malignancies are forms of cancer that begin in the cells of blood-forming tissue, such as the bone marrow, or in the cells of the immune system. Examples of haematologic malignancies are acute and chronic leukaemias, lymphomas, multiple myeloma, AML, non-Hodgkin lymphoma, and subtypes such as DLBCL, marginal zone, mantle zone, and follicular, Hodgkin lymphoma, and myelodysplastic syndromes.

[0382] In an embodiment the malignancy is an Acute Myeloid Leukaemia (AML) or AML derived cancer.

[0383] In one embodiment, the disease is a CD7+CD33+ haematological malignancy. The term "CD7+CD33+ haematological malignancy" refers to a haematological malignancy characterized by the expression of both CD7 and CD33 on the surface of the malignant cells (e.g., a haematological malignancy that over expresses CD33 and / or CD7 on their cell surface and / or that express CD33 and / or CD7 at levels considered acceptable for therapy with the antibody or antigen binding fragments thereof that specifically binds to CD7 and CD33).

[0384] CD7+CD33+ haematological malignancies include, but are not limited to, acute myeloid leukemia (AML), a myelodysplastic syndrome, a T-cell acute lymphoblastic leukemia, and a blastic plasmacytoid dendritic cell neoplasm (BPDCN).

[0385] Preferably, the dual targeting therapy described herein will provide a benefit to the treatment of a CD7+CD33+ haematological malignancy in a subject in need thereof. For example, the dual targeting therapy may have an additive or synergistic effect on the treatment of AML in a subject in need thereof. A dual targeting therapy is defined as affording an “additive effect”, “synergistic effect” or a “synergistic treatment” if the effect is therapeutically superior, as measured by, for example, the extent of the response (e.g. apoptosis or cell viability), the response rate, the time to disease progression or the survival period, to that achievable on dosing one or other of the components of the dual targeting therapy at its conventional dose. For example, the effect of the dual targeting therapy is additive if the effect is therapeutically superior to the effect achievable with an antibody or antigen binding fragments thereof that specifically binds to CD33 or CD7 alone. For example, the effect of the combination treatment may be synergistic if the effect of the combination treatment supersedes the effect of the individual treatments added together. Further, the effect of the combination is beneficial (e.g. additive or synergistic) if a beneficial effect is obtained in a group of subjects that does not respond (or responds poorly) to a cell-inhibiting agent that specifically binds to CD33 alone or a cell-inhibiting agent that specifically binds to CD7 alone. In addition, the effect of the combination treatment is defined as affording a benefit (e.g. additive or synergistic effect) if one of the components is dosed at its conventional dose and the other component is dosed at a reduced dose and the therapeutic effect, as measured by, for example, the extent of the response, the response rate, the time to disease progression or the survival period, is equivalent to or better than that achievable on dosing conventional amounts of either one of the components of the combination treatment.

[0386] As used herein, "killing of a target cell" relates for example to an inhibition of protein synthesis, for example such that cell viability is reduced, or an induction of apoptosis resulting in elimination or death of target cells. Assays to determine cell killing and apoptosis are well known in the art. Cytotoxicity assays assess the number of live and dead cells in a population after treatment with a pharmacological substance (e.g. an LDH cytotoxicity assay, or a live-dead cell assay). Apoptosis assays assess how cells are dying by measuring markers that are activated upon cell death (e.g. a Phosphatidylserine (PS) expos ure / ann exin V binding assay, a caspase activation assay, a DNA fragmentation assay, a GSH / GSSG determination, a LDH cytotoxicity assay, a live-dead cell assay, or a non-caspase protease activation assay).

[0387] As used herein "inhibit the cell growth” (e.g., referring to target cells) refers to any measurable decrease in the growth or proliferation of a target cell when contacted with the antibody or antigen binding fragments thereof according to the present invention as compared to the growth of the same cell not in contact with the antibody or antigen binding fragments thereof according to the present disclosure, e.g., the inhibition of growth of a cell by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 100%. Assays to determine cell viability or proliferation are well known in the art. Cell viability assays assess how healthy the cells are by measuring markers of cellular activity (e.g. an ATP and ADP determination assay, a cell cycle assay, a cell proliferation assay, a cell viability assay, an LHD cytotoxicity assay, or a live-dead cell assay). Cell proliferation assays assess the growth rate of a cell population or to detect daughter cells in a growing population (e.g. a cell cycle assay, a cell proliferation assay, a cell viability assay, or a senescence assay).

[0388] As used herein, "CD33 expressing cell" and “CD33+ cell” refers to a cell with CD33 as surface antigen. As used herein, "CD7 expressing cell" and “CD7+ cell” refers to a cell with CD7 as surface antigen. As used herein, "CD33 and CD7 expressing cell" and “CD33+CD7+ cell” refers to a cell with both CD33 and CD7 as surface antigens.

[0389] As used herein "target cell" refers to a cell or cell-type characterized by the expression or overexpression of the target molecule CD7 and CD33. Any type of cell expressing CD7 and CD33 may be envisaged as a target cell for treatment with the antibody or antigen binding fragments thereof of the invention. In certain embodiments, the cell is a tumour cell, for example a tumour cell from a haematological malignancy, such as an AML cell.

[0390] The bispecific binding molecule, immunoconjugate, antibody drug conjugate may be used in combination with any other anti-cancer therapy. A binding molecule, immunoconjugate, antibody drug conjugate may be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated. Examples of treatments and therapies include, but are not limited to, chemotherapy, immunotherapy, surgery and / or radiation therapy. Such therapy or therapeutic agent includes for example an anticancer compound, such as a chemotherapy agent, biologic, cytokine, small molecule, CAR-T therapy or radiotherapy treatment.

[0391] Chemotherapy agents include alkylating agents, plant alkaloids, antimetabolites, anthracyclines, topoisomerase inhibitors and corticosteroids. For example, the chemotherapy can include vinorelbine, cisplatin, carboplatin, gemcitabine, paclitaxel, topotecan, docetaxel, irinotecan, pemetrexed, etoposide, or any combination thereof.

[0392] A biologic may be an antibody therapy, for example an antibody that targets a checkpoint inhibitor, such as PD-1 (e.g. Pembrolizumab, Nivolumab or Cemiplimab), PD-L1 (e.g. Atezolizumab, Avelumab or Durvalumab), PD-L2, LAG-3 (e.g. Relatlimab), Tim-3 or CTLA4 (e.g. Ipilimumab).

[0393] The small molecule therapy may be Pexidartinib.

[0394] The bispecific binding molecule, immunoconjugate, antibody drug conjugate of the invention may be combined in a pharmaceutical combination formulation, or dosing regimen as combination therapy, with a second compound having anti-cancer properties. The second compound of the pharmaceutical combination formulation or dosing regimen may have complementary activities to bispecific binding molecule, immunoconjugate, antibody drug conjugate of the combination such that they do not adversely affect each other.

[0395] The skilled person will be able to determine appropriate dosage regimens of the bispecific binding molecule or conjugates described herein. The amount of the bispecific binding molecule, conjugate or pharmaceutical composition described herein that is effective / active in the treatment of a particular disease or condition will depend on the nature of the disease or condition and can be determined by standard clinical techniques. In addition, in vitro or in vivo assays can optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the compositions will also depend on the route of administration, and the seriousness of the disease or disease, and should be decided according to the judgment of the practitioner and each patient's circumstances. Factors like age, body weight, sex, diet, time of administration, rate of excretion, condition of the host, drug combinations, reaction sensitivities and severity of the disease shall be taken into account.

[0396] As an example the bispecific binding molecule, conjugate or pharmaceutical composition may be provided at a dose of 0.1 mg / kg to 100mg / kg, 0.5 mg / kg to 100mg / kg, 1 mg / kg to 100mg / kg, 2 mg / kg to l OOmg / kg, 5 mg / kg to 100mg / kg, 10 mg / kg to 100mg / kg, 20 mg / kg to 100mg / kg, 0.1 mg / kg to 80mg / kg, 0.5 mg / kg to 80mg / kg, 1 mg / kg to 80mg / kg, 2 mg / kg to 80mg / kg, 5 mg / kg to 80mg / kg, 10 mg / kg to 80mg / kg, 20 mg / kg to 80mg / kg, 0.1 mg / kg to 60mg / kg, 0.5 mg / kg to 60mg / kg, 1 mg / kg to 60mg / kg, 2 mg / kg to 60mg / kg, 5 mg / kg to 60mg / kg, 10 mg / kg to 60mg / kg, 20 mg / kg to 60mg / kg, 0.1 mg / kg to 40mg / kg, 0.5 mg / kg to 40mg / kg, 1 mg / kg to 40mg / kg, 2 mg / kg to 40mg / kg, 5 mg / kg to 40mg / kg, 10 mg / kg to 40mg / kg, 20 mg / kg to 40mg / kg, 0.1 mg / kg to 30mg / kg, 0.5 mg / kg to 30mg / kg, 1 mg / kg to 30mg / kg, 2 mg / kg to 30mg / kg, 5 mg / kg to 30mg / kg, 10 mg / kg to 30mg / kg, 15 mg / kg to 30mg / kg, 20 mg / kg to 30mg / kg, 0.1 mg / kg to 20mg / kg, 0.5 mg / kg to 20mg / kg, 1 mg / kg to 20mg / kg, 2 mg / kg to 20mg / kg, 5 mg / kg to 20mg / kg, 10 mg / kg to 20mg / kg, or 15 mg / kg to 20mg / kg.

[0397] As an example the bispecific binding molecule, conjugate or pharmaceutical composition may be administered with a specific treatment schedule. The treatment cycle may be 1 day to 42 days 1 day to 35 days, 1 day to 28 days, 1 day to 21 days, 1 day to 14 days, 1 day to 7 days. Within the treatment cycle the bispecific binding molecule, conjugate or pharmaceutical composition may be administered to the subject. Th treatment cycle may comprise 1 dose, 2 doses, 3 doses, 4 doses, 5 doses, 6 doses, 7 doses, 8 doses, 9 doses, 10 doses, 11 doses, 12 doses. The treatment cycle may comprise 1 dose every 7 days, 2 doses every 7 days, 3 doses every 7 days. The treatment cycle may comprise 1 dose every 14 days, 2 doses every 14 days, 3 doses every 14 days, 4 doses every 14 days, 5 doses every 14 days, 6 doses every 14 days.

[0398] As will be appreciated by the skilled person, the terms “treating”, “treats” and “treatment” include both preventative and curative treatment of a condition, disease or disorder. These terms also include slowing, interrupting, controlling or stopping the progression of a condition, disease or disorder and preventing, curing, slowing, interrupting, controlling or stopping the symptoms of a condition, disease or disorder. As used herein, "treat", "treating" or "treatment" means inhibiting or relieving a disease or disease. For example, treatment can include a postponement of development of the symptoms associated with a disease or disease, and / or a reduction in the severity of such symptoms that will, or are expected, to develop with said disease. The terms include ameliorating existing symptoms, preventing additional symptoms, and ameliorating or preventing the underlying causes of such symptoms. Thus, the terms denote that a beneficial result is being conferred on at least some of the mammals, e.g., canine patients, being treated. Many medical treatments are effective for some, but not all, patients that undergo the treatment. In an embodiment the present bispecific binding molecule increases subject survival compared to vehicle control. In an embodiment the present bispecific binding molecule increases subject survival compared to standard therapy, e.g., Cytaribine.

[0399] The term "subject" or "patient" refers to an animal which is the object of treatment, observation, or experiment. By way of example only, a subject includes, but is not limited to, a mammal, including, but not limited to, a human or a non-human mammal, such as a non-human primate, murine, bovine, equine, canine, ovine, or feline.

[0400] As used herein, a "patient" is typically a human who is undergoing treatment for, or has been diagnosed as having, haematological malignancy, preferably a CD7+CD33+ haematological malignancy. In some embodiments, the antibody or antigen binding fragments thereof are administered to a patient in remission from CD7+CD33+ haematological malignancy, whereby the recurrence of the haematological malignancy is prevented or delayed. In some embodiments, the patient lacks detectable cells of the haematological malignancy.

[0401] Wherein the bispecific binding molecule, immunoconjugate or antibody drug conjugate is used in therapy it will be administered to the subject at a therapeutically effective dose. The term "therapeutically effective amount" refers to an amount of a drug effective to treat a disease or disorder in a mammal. In the case of cancer, the therapeutically effective amount of the drug may reduce the number of cancer cells; reduce the tumor size; inhibit (i.e., slow to some extent and preferably stop) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent and preferably stop) tumor metastasis; inhibit, to some extent, tumor growth; and / or relieve to some extent one or more of the symptoms associated with the cancer. To the extent the drug may prevent growth and / or kill existing cancer cells, it may be cytostatic and / or cytotoxic. For cancer therapy, efficacy can, for example, be measured by assessing the time to disease progression (TTP) and / or determining the response rate (RR).

[0402] The therapeutically effective amount of the bispecific antibody or antigen binding portion thereof or a pharmaceutical composition of the bispecific antibody or antigen binding portion thereof may be administered orally, topically, by inhalation, insufflation or parenterally. As the skilled person will appreciate, the bispecific antibody or antigen binding portion thereof, may be formulated, for example in a pharmaceutical composition as described herein. The bispecific antibody or antigen binding portion thereof be formulated for a particular administration route. For example, formulations suitable for oral administration include tablets, lozenges, hard or soft capsules, aqueous or oily suspensions, emulsions, dispersible powders or granules, syrups or elixirs. Suitable formulations for topical use include, for example, creams, ointments, gels, or aqueous or oily solutions or suspensions. Suitable formulations for inhalation include, for example, as a fine powder or a liquid aerosol. Suitable formulations for administration by insufflation include, for example, a fine powder. Suitable formulations for parenteral administration include, for example, a sterile aqueous or oily solution for intravenous, subcutaneous, intramuscular or intramuscular dosing or as a suppository for rectal dosing.

[0403] As will be appreciated by the skilled person, the therapeutically effective amount of the bispecific antibody or antigen binding portion thereof or pharmaceutical composition as described herein, will necessarily vary depending on the subject to be treated, the route of administration and the nature and severity of the disease to be treated.

[0404] As used herein, the term “effective amount” means an amount of antibody or antigen-binding fragment thereof of the invention, that when administered alone or in combination with an additional therapeutic agent to a cell, tissue, or subject, is effective to achieve the desired therapeutic or prophylactic effect under the conditions of administration. The term “effective amount” of a composition, as used herein, is intended to denote a non-lethal but sufficient amount of the composition to provide the desired effect. For example, in order to elicit a favourable response in a subject when treating a disorder or infection, the effective amount is the one which eliminates or diminishes the symptoms associated with the disorder. An effective amount may be determined by one of ordinary skill in the art, using routine experimentation.

[0405] In addition, in vitro or in vivo assays can optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the compositions will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. Factors like age, body weight, sex, diet, time of administration, rate of excretion, condition of the host, drug combinations, reaction sensitivities and severity of the disease shall be taken into account.

[0406] Typically, the amount is at least about 0.01 % of the antibody or antigen-binding fragment thereof of the present invention by weight of the composition. When intended for oral administration, this amount can be varied to range from about 0.1 % to about 80% by weight of the composition. Preferred oral compositions can comprise from about 4% to about 50% of the antibody of the present invention by weight of the composition.

[0407] Compositions of the present invention can be prepared so that a parenteral dosage unit contains from about 0.01 % to about 2% by weight of the antibody of the present invention.

[0408] For administration by injection, the composition can comprise from about typically about 0.1 mg / kg to about 250 mg / kg of the subject’s body weight, for example, between about 0.1 mg / kg and about 20 mg / kg of the animal's body weight, for example about 1 mg / kg to about 10 mg / kg of the animal's body weight. In one embodiment, the composition is administered at a dose of about 1 to 30 mg / kg, e.g., about 5 to 25 mg / kg, about 10 to 20 mg / kg, about 1 to 5 mg / kg, or about 3 mg / kg. The dosing schedule can vary from e.g., once a week to once every 2, 3, or 4 weeks.

[0409] Methods

[0410] The bispecific binding molecules may also be used in various methods, for example in non- therapeutic methods.

[0411] An aspect of the invention relates to an in vitro, ex vivo or in vivo method of detecting CD33+ / CD7+ cells within a biological sample, comprising obtaining a biological sample contacting said biological sample with a bispecific binding molecule as described herein. The bispecific molecule may be used in a variety of diagnostic tests that comprise the detection of CD33+ / CD7+ cells, for example immunoassays for the detection of cancer. The immunoassays per se are well-known and any of the well-known immunoassays may be employed. That is, classifying the known immunoassays according to the reaction type, known immunoassays include sandwich immunoassays, competition immunoassays, agglutination immunoassays, Western blot and the like. Classifying the known immunoassays according to the label employed, known immunoassays include fluorescence immunoassays, enzyme immunoassays, radio immunoassays, biotin immunoassays and the like. Any of these immunoassays may be employed. Further, diagnosis may be attained by immunohistostaining. In cases where a labelled antibody antigen-binding fragment thereof or binding molecule is used in the immunoassay, the methods per se for labelling an antibody are well-known, and any of the well-known methods may be employed.

[0412] The term "detecting" is used herein in the broadest sense to include both qualitative and quantitative measurements of a target molecule. Detecting includes identifying the mere presence of the target molecule in a sample as well as determining whether the target molecule is present in the sample at detectable levels. Detecting may be direct or indirect.

[0413] The bispecific binding molecule, for use in the method of detecting CD33+ / CD7+ cells, may further comprise an additional moiety that allows identification of the CD33+ / CD7+ cells. The bispecific binding molecule may comprise a label, for example a fluorescent molecule, p- galactosidase, luciferase molecules, secondary antibody, chemical dyes, fluorophores or a radioisotope. The method may the comprise a further step of detecting the fluorescent molecule, p-galactosidase, luciferase molecules, secondary antibody, chemical dyes, fluorophores or a radioisotope

[0414] An aspect of the invention relates to an in vitro, ex vivo or in vivo method of delivering a payload to CD33+ / CD7+ cells within a biological sample, comprising obtaining a biological sample contacting said biological sample with a bispecific binding molecule as described herein, wherein the bispecific molecule is conjugated to a payload.

[0415] The payload may be any payload as described herein, including but not limited to a cell killing agent, an immune-modulating payload, a macrophage class switching agent, a light activatable payload, or a detectable label.

[0416] An aspect of the invention relates to a method for reducing off target toxicity of a cancer treatment comprising administering to a subject a bispecific binding molecule, an immunoconjugate or an antibody drug conjugate as described herein. The biological sample may be a biological tissue sample, a biological fluid. A biological fluid may be, for example, blood, serum, lymph, urine, inflammatory exudate, cerebrospinal fluid, amniotic fluid, a tissue extract or homogenate, and the like.

[0417] Kits

[0418] An aspect of the invention relates to a kit comprising the bispecific binding molecule described herein and optionally instructions for use.

[0419] The kit may contain materials useful for the treatment of the disorders described above is provided. The kit comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, blister pack, etc. The containers may be formed from a variety of materials such as glass or plastic. The container holds the bispecific binding molecule, immunoconjugate or antibody drug conjugate of the invention which is effective for treating the condition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is an ADC. The label or package insert indicates that the composition is used for treating the condition of choice, such as cancer. The kit ma further contain a pharmaceutically- acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0420] Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. While the foregoing disclosure provides a general description of the subject matter encompassed within the scope of the present disclosure, including methods, as well as the best mode thereof, of making and using this disclosure, the following examples are provided to further enable those skilled in the art to practice this disclosure. However, those skilled in the art will appreciate that the specifics of these examples should not be read as limiting on the invention, the scope of which should be apprehended from the claims and equivalents thereof appended to this disclosure. Various further aspects and embodiments of the present disclosure will be apparent to those skilled in the art in view of the present disclosure.

[0421] All documents mentioned in this specification are incorporated herein by reference in their entirety, including references to gene accession numbers, scientific publications and references to patent publications.

[0422] "and / or" where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example "A and / or B" is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein. Unless context dictates otherwise, the descriptions and definitions of the features set out above are not limited to any particular aspect or embodiment of the invention and apply equally to all aspects and embodiments which are described.

[0423] The term “comprising” or “comprises” where used herein means including the component(s) specified but not to the exclusion of the presence of other components. The term “consisting essentially of’ or “consists essentially of’ means including the components specified but excluding other components except for materials present as impurities, unavoidable materials present as a result of processes used to provide the components and the like.

[0424] The term “consisting of’ or “consists of’ means including the components specified but excluding other components.

[0425] Whenever appropriate, depending upon the context, the use of the term “comprises” or “comprising” may also be taken to include the meaning “consists essentially of’ or “consisting essentially of’, and also may also be taken to include the meaning “consists of’ or “consisting of’.

[0426] The optional features set out herein may be used either individually or in combination with each other where appropriate and particularly in the combinations as set out in the accompanying claims. The optional features for each aspect or exemplary embodiment of the invention, as set out herein are also applicable to all other aspects or exemplary embodiments of the invention, where appropriate. In other words, the skilled person reading this specification should consider the optional features for each aspect or exemplary embodiment of the invention as interchangeable and combinable between different aspects and exemplary embodiments.

[0427] It should be understood that while the use of words such as “preferable”, “preferably”, “preferred” or “more preferred” in the description suggest that a feature so described may be desirable, it may nevertheless not be necessary and embodiments lacking such a feature may be contemplated as within the scope of the invention as defined in the appended claims. In relation to the claims, it is intended that when words such as “a,” “an,” or “at least one,” are used to preface a feature there is no intention to limit the claim to only one such feature unless specifically stated to the contrary in the claim.

[0428] The invention is further defined in the the following numbered aspects:

[0429] 1 . A bispecific binding molecule, comprising a first antigen binding domain that binds to CD7 and a second antigen binding domain that binds CD33, wherein said first antigen binding domain comprises ai) a heavy chain variable (VH) region comprising SEQ ID NO. 21 or a sequence comprising at least 90%, 92%, 95%, 97% or 99% sequence identity thereto, and aii) a light chain variable (VL) region comprising SEQ ID NO. 23 or a sequence comprising at least 80%, 85%, 90%, 92%, 95%, 97% or 99% sequence identity thereto, or bi) a heavy chain variable (VH) region comprising SEQ ID NO. 25 or a sequence comprising at least 90%, 92%, 95%, 97% or 99% sequence identity thereto, and bii) a light chain variable (VL) region comprising SEQ ID NO. 27 or a sequence comprising at least 80%, 85%, 90%, 92%, 95%, 97% or 99% sequence identity thereto, or ci) a heavy chain variable (VH) region comprising SEQ ID NO. 29 or a sequence comprising at least 90%, 92%, 95%, 97% or 99% sequence identity thereto, and cii) a light chain variable (VL) region comprising SEQ ID NO. 31 or a sequence comprising at least 80%, 85%, 90%, 92%, 95%, 97% or 99% sequence identity thereto, or di) a heavy chain variable (VH) region comprising SEQ ID NO. 33 or a sequence comprising at least 90%, 92%, 95%, 97% or 99% sequence identity thereto, and dii) a light chain variable (VL) region comprising SEQ ID NO. 35 or a sequence comprising at least 80%, 85%, 90%, 92%, 95%, 97% or 99% sequence identity thereto, or ei) a heavy chain variable (VH) region comprising SEQ ID NO. 37 or a sequence comprising at least 90%, 92%, 95%, 97% or 99% sequence identity thereto, and eii) a light chain variable (VL) region comprising SEQ ID NO. 39 or a sequence comprising at least 80%, 85%, 90%, 92%, 95%, 97% or 99% sequence identity thereto.

[0430] 2. The bispecific binding molecule of aspect 1 , wherein the bispecific antibody binding molecule is capable of targeting cells expressing CD7+CD33+, and wherein CD33 is expressed either at a high or a low level.

[0431] 3. The bispecific binding molecule of aspects 1 or 2, wherein the first antigen binding domain comprises ai) a heavy chain variable (VH) region comprising SEQ ID NO. 21 or a sequence comprising between 1 and 19 amino acid differences thereto, and aii) a light chain variable (VL) region comprising SEQ ID NO. 23 or a sequence comprising between 1 and 21 amino acid differences thereto, or bi) a heavy chain variable (VH) region comprising SEQ ID NO. 25 or a sequence comprising between 1 and 19 amino acid differences thereto, and bii) a light chain variable (VL) region comprising SEQ ID NO. 27 or a sequence comprising between 1 and 21 amino acid differences thereto, or ci) a heavy chain variable (VH) region comprising SEQ ID NO. 29 or a sequence comprising between 1 and 19 amino acid differences thereto, and cii) a light chain variable (VL) region comprising SEQ ID NO. 31 or a sequence comprising between 1 and 21 amino acid differences thereto, or di) a heavy chain variable (VH) region comprising SEQ ID NO. 33 or a sequence comprising between 1 and 19 amino acid differences thereto, and dii) a light chain variable (VL) region comprising SEQ ID NO. 35 or a sequence comprising between 1 and 21 amino acid differences thereto, or ei) a heavy chain variable (VH) region comprising SEQ ID NO. 37 or a sequence comprising between 1 and 19 amino acid differences thereto, and eii) a light chain variable (VL) region comprising SEQ ID NO. 39 or a sequence comprising between 1 and 21 amino acid differences thereto.

[0432] 4. The bispecific binding molecule according to any preceding aspect, wherein the first antigen binding domain comprises i) a heavy chain variable (VH) region comprising a sequence selected from SEQ ID NO.

[0433] 21 , SEQ ID NO. 25, SEQ ID NO. 29, SEQ ID NO. 33, or SEQ ID NO. 37 and ii) a light chain variable (VL) region comprising a sequence selected from SEQ ID NO. 23, SEQ ID NO. 27, SEQ ID NO. 31 , SEQ ID NO. 35, or SEQ ID NO. 39.

[0434] 5. The bispecific binding molecule according to any preceding aspect wherein the first antigen binding domain comprises i) a heavy chain comprising SEQ ID NO. 22 or a sequence comprising at least 70%, 80%, 85%, 90%, 95% or 99% sequence identity thereto, and ii) a light chain comprising SEQ ID NO. 24 or a sequence comprising at least 70%, 80%, 85%, 90%, 95% or 99% sequence identity thereto.

[0435] 6. The bispecific binding molecule according to any preceding aspect, wherein the first antigen binding domain comprises i) a heavy chain comprising a sequence selected from SEQ ID NO. 22, SEQ ID NO. 26, SEQ ID NO. 30, SEQ ID NO. 34, or SEQ ID NO. 38 and ii) a light chain variable (VL) region comprising a sequence selected from SEQ ID NO. 24, SEQ ID NO. 28, SEQ ID NO. 32, SEQ ID NO. 36, or SEQ ID NO. 40.

[0436] 7. The bispecific binding molecule according to any preceding aspect, wherein the second antigen binding domain comprises i) a heavy chain variable (VH) region comprising a HC CDR1 comprising SEQ ID NO. 2, a HC CDR2 comprising SEQ ID NO. 3, a HC CDR3 comprising SEQ ID NO. 4, and ii) a light chain variable (VL) region comprising a LC CDR1 comprising SEQ ID NO. 7, a LC CDR2 comprising SEQ ID NO. 8, a LC CDR3 comprising SEQ ID NO. 9.

[0437] 8. The bispecific binding molecule according to any preceding aspect, wherein the second antigen binding domain comprises i) a heavy chain variable (VH) region comprising SEQ ID NO. 1 and ii) a light chain variable (VL) region comprising SEQ ID NO. 6.

[0438] 9. The bispecific binding molecule of any preceding aspect, wherein the first antigen binding domain has a binding affinity (KD) to CD7 of between 5 x10-10M and 2 x10-7M,

[0439] 10. The bispecific binding molecule of any preceding aspect, wherein the second antigen binding domain has a binding affinity (KD) to CD33 of between 1 .5 x10-10M and 3 x10-8M. 11. The bispecific binding molecule of any preceding aspect, wherein said antibody or antigen binding fragment is capable of inducing CD33 and / or CD7 receptor mediated internalization into a CD33+ and / or CD7+ cell.

[0440] 12. The bispecific binding molecule of any preceding aspect, wherein said antibody or antigen binding fragment is capable of selectively targeting cells expressing CD7+ and CD33+, compared to cells expressing CD7+ or cells expressing CD33+.

[0441] 13. The bispecific binding molecule of any preceding aspect, wherein said first or second antigen binding domain comprises a Fab, Fab’, F(ab)2, scFv, (scFv), heavy chain only antibody, single domain antibody, nanobody,

[0442] 14. The bispecific binding molecule of any preceding aspect, wherein the first and second antigen binding domain are attached via a linker.

[0443] 15. The bispecific binding molecule of any preceding aspect, wherein the first and second antigen binding domain are conjugated together via click chemistry.

[0444] 16. The bispecific binding molecule of aspect 15, wherein the click chemistry used to conjugate together the first and second antigen binding domain is selected from a thiol-ene reaction, a copper catalyzed azide-alkyne cycloaddition (CuAAC) reaction, a strained-promoted azide-alkyne click chemistry (SPAAC) reaction and / or an inverse electron demand Diels-Alder (iEDDA) reaction, for example via an inverse electron demand Diels-Alder (iEDDA) reaction.

[0445] 17. The bispecific binding molecule of any preceding aspect, wherein said antibody or antigen binding fragment is conjugated to a payload.

[0446] 18. The bispecific binding molecule of any preceding aspect, wherein the payload is a cell killing agent, an immune-modulating payload, a macrophage class switching agent or a light activatable payload.

[0447] 19. The bispecific binding molecule of any preceding aspect, wherein the immune-modulating payload is a STING agonist or a toll-like receptor agonist.

[0448] 20. The bispecific binding molecule of aspect 18, wherein the cell killing agent comprises a cytotoxin.

[0449] 21 . The bispecific binding molecule of aspect 20, wherein said cytotoxin is selected from: i) a peptide toxin; or ii) a chemical toxin.

[0450] 22. The bispecific binding molecule of any one of aspects 17 to 21 , wherein the first antigen binding domain has a binding affinity (KD) to CD7 of between 6.5 x10-10M and 2 x10-7M.

[0451] 23. The bispecific binding molecule of any preceding aspect, further comprising a linker for linking the payload to the bispecific antibody or antigen binding fragment.

[0452] 24. The bispecific binding molecule of aspect 23, wherein the linker is attached to the bispecific antibody or antigen binding fragment via a non-naturally occurring amino acid residue present in the bispecific antibody or antigen binding fragment. 25. The bispecific binding molecule of aspect 24, wherein the non-naturally occurring amino acid residue is selected from a cysteine residue, a lysine residue, a histidine residue, a tyrosine residue, formylglycine residue.

[0453] 26. The bispecific binding molecule of any one of aspects 30 to 32, wherein the linker is selected from one or more of a cleavable linker, a non-cleavable linker, a pH sensitive linker, a redox sensitive linker.

[0454] 27. The bispecific binding molecule of any preceding aspect, further comprising an additional moiety selected from a half-life extending moiety and / or a label.

[0455] 28. The bispecific binding molecule of aspect 27, wherein the half-life extending moiety is selected from serum albumin protein, a binding molecule that binds serum albumin, an Fc domain and / or a modified Fc domain.

[0456] 29. The bispecific binding molecule of any preceding aspect, further comprising one or more additional binding domains.

[0457] 30. An isolated nucleic acid molecule encoding the bispecific antibody or antigen binding fragment of any one of aspects 1 to 29.

[0458] 31 . A vector encoding the isolated nucleic acid of aspect 30.

[0459] 32. A host cell comprising the nucleic acid of aspect 30, or the vector of aspect 31 .

[0460] 33. An immunoconjugate comprising the bispecific binding molecule of any of aspects 1 to 29.

[0461] 34. A pharmaceutical composition comprising the bispecific binding molecule of any one of aspects 1 to 29 and optionally a pharmaceutically acceptable excipient, and / or adjuvant.

[0462] 35. The bispecific binding molecule according to any one of aspects 1 to 29 for use in the treatment of a malignancy.

[0463] 36. A method of treating or preventing a malignancy comprising administering a therapeutically effective amount of the bispecific antibody or antigen binding fragment of any one of aspects 1 to 29.

[0464] 37. The use of the bispecific binding molecule of any one of aspects 1 to 29 for the manufacture of a medicament for the treatment of a malignancy.

[0465] 38. The bispecific binding molecule for use according to aspect 35, the method according to aspect 36 of the use according to aspect 37, wherein the malignancy is selected from one of the following cancers: haematological cancers or Acute Myeloid Leukaemia.

[0466] 39. The bispecific binding molecule for use according to aspect 40, the method according to aspect 36 of the use according to aspect 37, wherein the malignancy is an Acute Myeloid Leukaemia (AML)or AML derived cancer.

[0467] 40. An in vitro, ex vivo or in vivo method of detecting CD33+ / CD7+ cells within a biological sample, comprising obtaining a biological sample contacting said biological sample with a bispecific binding molecule according to any one of aspects 1 to 29. 41 . An in vitro, ex vivo or in vivo method of delivering a payload to CD33+ / CD7+ cells within a biological sample, comprising

[0468] Obtaining or providing a biological sample contacting said biological sample with a bispecific binding molecule according to any one of aspect 1 to 29.

[0469] 42. A method for reducing off target toxicity of a cancer treatment comprising administering to a subject a bispecific binding molecule according to any one of aspects 1 to 29.

[0470] 43. A kit comprising the bispecific binding molecule of any one of aspects 1 to 29 and optionally instructions for use.

[0471] The invention is further illustrated in the following non-limiting examples.

[0472] Features of the use of the present invention are described in further detail in relation to the abovementioned aspects of the invention, in the examples below.

[0473] The described and illustrated examples are to be considered as illustrative and not restrictive in character, it being understood that only the preferred embodiments have been shown and described and that all changes and modifications that come within the scope of the inventions as defined in the claims are desired to be protected.

[0474] EXAMPLES

[0475] Example 1 - Cell Cytotoxicity

[0476] Example 1 demonstrates that the selected antibody sequences show greater cell selectivity for target CD33+ / CD7+ cells compared to molecules with differing CD7 and CD33 sequences targeting sequences. Further when used in ADC format the selected antibody sequences show greater cell selectivity and potency for target CD33+ / CD7+ cells versus non-target cells when compared to larger panel of bispecific ADCs with differing CD7 and CD33 sequences.

[0477] Reagents

[0478] Cell Lines (KASUMI-3, HNT-34, JUKRAT, MV4-11 , SHI-1 , DND-39) DSMZ

[0479] BVX01-a0032-AB4A (antiCD33 x antiCD7 Bi-Fab ADC) In house

[0480] BVX01-a0054-AB4A (antiCD33 x antiCD7 Bi-Fab ADC) In house

[0481] BVX01-a0057-AB4A (antiCD33 x antiCD7 Bi-Fab ADC) In house

[0482] BVX01-a0059-AB4A (antiCD33 x antiCD7 Bi-Fab ADC) In house

[0483] BVX01-a0062-AB4A (antiCD33 x antiCD7 Bi-Fab ADC) In house

[0484] BVX01-a0063-AB4A (antiCD33 x antiCD7 Bi-Fab ADC) In house

[0485] BVX01-a0090-AB4A (antiCD33 x antiCD7 Bi-Fab ADC) In house

[0486] BVX01-a0092-AB4A (antiCD33 x antiCD7 Bi-Fab ADC) In house

[0487] BVX01-b0015-AB6A (antiCD33 ScFvx antiCD7 Fab ADC) In house BVX01-b0037-AB6A (antiCD33 ScFvx antiCD7 Fab ADC) In house

[0488] BVX01-b0040-AB6A (antiCD33 ScFvx antiCD7 Fab ADC) In house

[0489] BVX01-b0042-AB6A (antiCD33 ScFvx antiCD7 Fab ADC) In house

[0490] BVX01-b0041-AB6A (antiCD33 ScFvx antiCD7 Fab ADC) In house

[0491] BVX01-b0069-AB6A (antiCD33 ScFvx antiCD7 Fab ADC) In house

[0492] BVX01-b0083-AB6A (antiCD33 ScFvx antiCD7 Fab ADC) In house

[0493] BVX010158 (antiCD33 Fab x antiCD7 ScFv ADC) In house

[0494] BVX010172 (antiCD33 Fab x antiCD7 ScFv ADC) In house

[0495] BVX010173 (antiCD33 Fab x antiCD7 ScFv ADC) In house

[0496] BVX010175 (antiCD33 Fab x antiCD7 ScFv ADC) In house

[0497] BVX010176 (antiCD33 Fab x antiCD7 ScFv ADC) In house

[0498] BVX010180 (antiCD33 Fab x antiCD7 ScFv ADC) In house

[0499] Clear bottom 96-well plates CytoOne®, Non-Treated (#CC7672-7596) STARLABS Disposable PS Reservoirs-StarTub PS (#E2310-1010) STARLABS

[0500] Cell Proliferation Reagent WST-1 (#ab155902) Abeam

[0501] 96-Well Round Bottom 2mL Polypropylene Deep Well Plate (#AXYPDW20CS) SLS

[0502] RPMI-1640 medium (#21875059) Life Technologies

[0503] Fetal Bovine Serum, Heat inactivated (#11533387) Life Technologies

[0504] Description of the molecules

[0505] BVX01-a0032-AB4A comprises a CD7 binding arm comprising SEQ ID NO. 21 and 23 and a CD33 binding arm comprising SEQ ID NO. 1 and 6.

[0506] BVX01-a0059-AB4A comprises a CD7 binding arm comprising SEQ ID NO. 25 and 27 and a CD33 binding arm comprising SEQ ID NO. 1 and 6.

[0507] BVX01-a0054-AB4A comprises a CD7 binding arm comprising SEQ ID NO. 29 and 31 and a CD33 binding arm comprising SEQ ID NO. 1 and 6.

[0508] BVX01-a0090-AB4A comprises a CD7 binding arm comprising SEQ ID NO. 33 and 35 and a CD33 binding arm comprising SEQ ID NO. 1 and 6.

[0509] BVX01-a0092-AB4A comprises a CD7 binding arm comprising SEQ ID NO. 37 and 39 and a

[0510] CD33 binding arm comprising SEQ ID NO. 1 and 6.

[0511] BVX01-a0057-AB4A comprises a CD33 binding arm comprising SEQ ID NO. 1 and 6 and a humanised CD7 binding arm comprising different humanisation mutations than the lead panel.

[0512] BVX01-a0062-AB4A comprises a CD33 binding arm comprising SEQ ID NO. 1 and 6 and a humanised CD7 binding arm comprising different humanisation mutations than the lead panel.

[0513] BVX01-a0063-AB4A comprises a CD33 binding arm comprising SEQ ID NO. 1 and 6 and a humanised CD7 binding arm comprising different humanisation mutations than the lead panel.

[0514] BVX01-b0015-AB6A - this is an asymmetric molecule comprising an antiCD33 ScFv binding arm and an antiCD7 Fab binding arm. BVX01-b0037-AB6A this is an asymmetric molecule comprising an antiCD33 ScFv binding arm and an antiCD7 Fab binding arm.

[0515] BVX01-b0040-AB6A this is an asymmetric molecule comprising an antiCD33 ScFv binding arm and an antiCD7 Fab binding arm.

[0516] BVX01-b0042-AB6A this is an asymmetric molecule comprising an antiCD33 ScFv binding arm and an antiCD7 Fab binding arm.

[0517] BVX01-b0041-AB6A this is an asymmetric molecule comprising an antiCD33 ScFv binding arm and an antiCD7 Fab binding arm.

[0518] BVX01-b0069-AB6A this is an asymmetric molecule comprising an antiCD33 ScFv binding arm and an antiCD7 Fab binding arm.

[0519] BVX01-b0083-AB6A this is an asymmetric molecule comprising an antiCD33 ScFv binding arm and an antiCD7 Fab binding arm.

[0520] BVX010158 this is an asymmetric molecule comprising an antiCD33 Fab binding arm and an antiCD7 ScFv binding arm.

[0521] BVX010172 (antiCD33 Fab x antiCD7 ScFv ADC) this is an asymmetric molecule comprising an antiCD33 Fab binding arm and an antiCD7 ScFv binding arm.

[0522] BVX010173 (antiCD33 Fab x antiCD7 ScFv ADC) this is an asymmetric molecule comprising an antiCD33 Fab binding arm and an antiCD7 ScFv binding arm.

[0523] BVX010175 (antiCD33 Fab x antiCD7 ScFv ADC) this is an asymmetric molecule comprising an antiCD33 Fab binding arm and an antiCD7 ScFv binding arm.

[0524] BVX010176 (antiCD33 Fab x antiCD7 ScFv ADC) this is an asymmetric molecule comprising an antiCD33 Fab binding arm and an antiCD7 ScFv binding arm.

[0525] BVX010180 (antiCD33 Fab x antiCD7 ScFv ADC) this is an asymmetric molecule comprising an antiCD33 Fab binding arm and an antiCD7 ScFv binding arm.

[0526] Methods

[0527] Fabs were modified to permit Bi-Fab formation by bioorthogonal reactive partners. On formation of the Bi-Fab, it was conjugated with mcMMAF average DAR 4. Asymmetrical ADC molecules were reduced with DTT and conjugated with mcMMAF, average DAR 4 or DAR 6. Cell lines were harvested, counted and the volume required to seed 20,000 (KASUMI-3, MV4-11 , SHI-1), 10,000 (HNT-34) and 5,000 (JURKAT, DND-39) cells per well in 50pl media was calculated for a 96-well plate. A 9-point dose response of ADC concentration was prepared in assay media (RPMI-1640, 10% FBS) at 2xthe final concentration with a top final concentration of either 30nM or 100nM. 50pl of each dose was pipetted across duplicate wells in a 96 well plate and a separate plate was prepared for each cell line tested. 10OpI of assay media was pipetted in the blank control and 50pl in the cell-only control wells; the plates were incubated at 37°C, 5% CO2 for 96 hours. After 96 hours incubation, 10pl of WST-1 reagent was added per well and after 3 hours incubation at 37°C, 5% CO2 the absorbance read at 440nm and 620nm. The data for each reading was plotted in GraphPad PRISM and the IC50 values recorded. The CD33+CD7+ cell lines KASUMI-3 and HNT-34 represent “target cancer cells” (KASUMI-3 CD33+HIGHCD7+HIGH; HNT-34 CD33+LOWCD7+HIGH); the single positive cell lines (JURKAT CD33-CD7+; SHI-1 CD33+CD7- and MV4-11 CD33+CD7-) and the double negative cell line (DND-39 CD33-CD7-) represent “non-target cells”.

[0528] Cell Line Profiling

[0529] Cell Lines were harvested and washed 2x with PBS before incubating for 1 hour on ice with anti- CD33 antibody (Absolute antibody #Ab00283-1 .1) or anti-CD7 antibody (Abeam #ab213014) diluted in 0.1 % BSA PBS 1 :100. Following the manufacturer’s instructions, the QIFI-kit (Agilent #K0078) was used to determine absolute receptor number on the cells. In brief, 10Oul of set up and calibration beads were stained alongside the cells. Beads and cells were washed 2x with 0.1 % BSA PBS and then incubated for 45 minutes on ice with Goat anti-mouse IgG-FITC antibody before washing 2x in PBS. Cells were then run on the MACSQuant 16 flow cytometer and the specific antibody binding capacity (SABC) (absolute receptor number) was calculated as per the manufacturer’s instructions and given in Antibody-Binding Capacity (ABC) units.

[0530] Table 1 - Cell line profiling

[0531] Results

[0532] Figures 1 to 4 show that overall the IC50 values indicated that certain bispecific ADCs showed greater potency and cell selectivity to target CD33+HIGH / CD7+ KASUMI-3 and CD33+LOW / CD7+ HNT-34 cells compared to the larger panel of bispecific ADCs screened, although all bispecific ADCs showed some degree of selectivity for target versus non-target cells. The cell selectivity of all clones was compared and together with other data (developability) the following bispecific ADCs were selected as the lead panel based on the best selectivity: BVX01- a0032-AB4A and BVX01-a0059-AB4A, BVX01-a0054-AB4A, BVX01-a0090-AB4A and BVX01- a0092-AB4A.

[0533] Table 2 - IC50

[0534]

[0535] Example 2 - Xenograft Mouse Efficacy

[0536] Example 2 demonstrates the selected lead bispecific ADCs (as selected in Example 1) show good efficacy in vivo using target HNT-34 (CD33+LOW / CD7+) xenograft model.

[0537] Reagents

[0538] HNT-34 (CD33+LOW / CD7+) cell line DSMZ

[0539] BVX01-a0032-AB4A (antiCD33 x antiCD7 Bi-Fab ADC) In house

[0540] BVX01-a0059-AB4A (antiCD33 x antiCD7 Bi-Fab ADC) In house SCID mice Sygnature Discovery

[0541] Cytarabine Sygnature Discovery

[0542] Methods

[0543] The aim of the study is to assess the efficacy of TA-1 (BVX132), TA-2 (BVX159) and Cytarabine in the HNT34 model of acute myeloid leukemic grown in CB17 SCID mice. Animals were randomised on to study when average tumour volume reached approximately 0.2cm3.

[0544] All work was carried out under UK Home Office legislation by Sygnature Discovery contract research organisation. Female SCID mice (CB17 SCID) aged 6 weeks old purchased from Charles River, UK were implanted subcutaneously (s.c.) with 0.1 ml of the human acute myeloid leukaemia cell line HNT-34 (CD33+LOW / CD7+) at 5x106cells per mouse in RPMI 1640 mixed 1 :1 with matrigel. Animals were randomised into groups of either 12 (vehicle and cytarabine) or 9 (test article dose groups) once average tumour volume reached approximately 0.2 cm3. Animals were dosed intravenously (i.v) with bispecific ADC formulated in PBS and vehicle (PBS, dosed twice weekly) for 4 weeks or intraperitoneally (i.p) with cytarabine (20mg / kg) 5 days on, 2 days off for 4 weeks. Dosing level and frequency of bispecific ADCs were as described in Figure 5 legend. Tumours were measured twice weekly by caliper and the volume of tumours calculated. Fabs were modified to permit Bi-Fab formation by bioorthogonal reactive partners. On formation of the Bi-Fab, it was conjugated with mcMMAF average DAR 4.

[0545] Animals were weighed at the same time as tumour measure and health checked daily.

[0546] Table 3 - Study dosing schedule

[0547] Table 4 - Summary of % TGI (tumour growth inhibition) or tumour regression compared to vehicle on Day 28 Tumour growth inhibition (%TGI) from the start of treatment was assessed by comparison of the geometric mean change in tumour volume for the control and treated groups. Tumour regression was calculated as the percentage reduction in tumour volume from baseline value % Regression = (1 - RTV) x 100%, where RTV is the geometric mean relative tumour volume. Statistical significance was evaluated using a one-tailed t-test.

[0548] Results

[0549] Figure 5 shows both bispecific ADCs BVX01-a0032-AB4A ADC and BVX01-a0059-AB4A ADC showed good efficacy in vivo, leading to a highly significant reduction in tumour volume compared to vehicle control. Both molecules share the same antiCD33_1 Fab as well as all CDRs for antiCD7 Fab. They differ in a number of framework residues as a result of humanisation.

[0550] Example 3 - Humanised mouse model

[0551] Example 3 demonstrates that BVX001-a0010-AB4A bispecific ADC shows improved safety versus Mylotarg® in a humanised mouse model.

[0552] BVX01-a0010-AB4A Bi-Fab ADC is a non-humanised variant of BVX01-a0032-AB4A and was used as a proof-of-concept molecule. Here, the antiCD33 Fab as well as methods for click chemistry and stochastic toxin conjugation are identical to BVX01-a0032-AB4A. The anti-CD7 Fab moiety subsequently was humanised (see also lead panel sequence alignment) in frameworks only. All CDRs of the anti-CD7 Fab are identical between humanised and nonhumanised variant. In terms of target binding, cell-selectivity, and cell cytotoxic potency, both BVX001-a0010-AB4A Bi-Fab ADC and BVX01-a0032-AB4A are comparable.

[0553] Reagents

[0554] PBS vehicle control Champions Oncology

[0555] BVX01-a0010-AB4A (antiCD33 x antiCD7 Bi-Fab ADC) In house Mylotarg® (Gemtuzumab Ozogamicin; GO) Sourced from Clinigen

[0556] Immune-compromised mice Champions Oncology-TransCure

[0557] Bioservices

[0558] Method - humanised mouse model

[0559] The Transcure Bioservices (transcurebioservices.com) CD34+ humanised mouse model was used. Mice were prepared and treated by the CRO to establish a mouse reconstituted with a fully human immune system. The reconstituted mice were treated on day 0 with vehicle control, Mylotarg® (GO, 0.3 mg i kg) or BVX01-a0010-AB4A (0.3mg i kg or 3mg i kg). Bone marrow and blood samples were collected at day 7 and day 14 for all mice. Samples were analysed by flow cytometry for the presence or absence of different markers which represent different cell lineages, including CD7+ T-cells and NK cells, CD33+ myeloid cells, CD11 b+ myeloid subset and CD11 b+CD15+ neutrophils. Results

[0560] Compared to Mylotarg® (GO), BVX01-a0010-AB4A Bi-Fab ADC showed significantly improved safety in this humanised mouse model. Mylotarg® (GO) treatment led to a significant reduction in the % of CD33+ myeloid cells compared to control, whereas this was not observed for BVX01 - a0010-AB4A Bi-Fab ADC treated mice. The number of neutrophils was also significantly reduced after Mylotarg® (GO) treatment whereas this was not observed for BVX01 -a0010-AB4A Bi-Fab ADC treated mice - neutrophil levels here remained comparable to the vehicle group. This data (see Figure 6) indicates that the Bi-Fab format of an ADC is tolerated well in this humanised mouse model and demonstrates the potential for less toxicity towards healthy human bone marrow and blood cells in the clinic.

[0561] Example 4 - Developability assessment

[0562] Developability assessment evaluates the drug-like properties of lead candidates at the early stage (i.e. discovery), for the potential of being successfully developed into a stable, manufacturable, safe, and efficacious drug product. A set of in vitro assays with predictive potential were used to select a panel of leads to take forward.

[0563] Table 5 - Set of Developability parameters used to assess BVX001 lead panel.

[0564] The lead panel of bispecific ADCs was assessed against a comprehensive set of biological, biochemical and bio-physical parameters and scored using a grey-scale to identify those clones with the greatest potential to be developable.

[0565] Here, light grey reflects adherence of a particular clone to a developability parameter; dark grey signifies a deviation from that parameter requiring particular attention going forward whereas black identifies a liability that renders this particular clone not developable. Table 6 below highlights some clones with all light grey, some with dark grey and others with black developability parameters. Note, that not all parameters need to be assessed if a liability is identified early. Clones with black were discounted for further assessment and dropped off the lead panel. BVX01-a0032-AB4A and BVX01-a0059-AB4A were the most promising clones according to their developability profile.

[0566] Table 6 - Summary of Lead Panel clones and their developability assessment against a set of criteria. Light grey: compliance; dark grey: some deviation; black: liability

[0567] Example 5 - Payload Deconjugation Assessment

[0568] Reagents

[0569] BVX01-a0032-AB4A (antiCD33 x antiCD7 Bi-Fab ADC) In house BVX01-a0054-AB4A (antiCD33 x antiCD7 Bi-Fab ADC) In house BVX01-a0059-AB4A (antiCD33 x antiCD7 Bi-Fab ADC) In house BVX01-a0092-AB4A (antiCD33 x antiCD7 Bi-Fab ADC) In house HSA (#SRP6182) Sigma-Aldrich

[0570] 0.5 ml sterile Eppendorf tubes (#10401203) Fisher Scientific

[0571] Parafilm® (#P7543) Sigma-Aldrich

[0572] Methods

[0573] Sample preparation

[0574] Where possible, samples were diluted to 1 mg / ml with 1 x PBS and 180 pL was pipetted into 0.5 mL sterile Eppendorf tubes. Tubes were sealed with Parafilm to ensure no evaporation occurred. The T=0 sample was snap frozen immediately in liquid nitrogen and stored at -80 °C until required. The T=3 and T=5 samples were incubated at 40 °C for 3 and 5 days, respectively. Following incubation, all samples were frozen in liquid nitrogen and stored at -80 °C.

[0575] Reverse phase chromatography and DAR calculation

[0576] Following defrost of the timepoint samples, HPLC-RP was used as a method to investigate stability of the samples. HPLC vials were charged with 100 pg of sample at 1 mg / mL and a 10 pl sample was analysed by RP. The UV trace was monitored at 214 nm, 252 nm and 280 nm.

[0577] The average DAR was calculated by comparison of reverse phase spectra at T=0 / 3 / 5 days to the spectrum of a sample of reduced Bi-Fab prior to the addition of mcMMAF Figure 8. The conjugation of mcMMAF to a protein chain causes an increase in the retention time of a particular chain. After addition of mcMMAF, comparison of a shifted peak area to any unshifted peak area was used to calculate the % of a particular chain that had been conjugated with payload and therefore allowed the calculation of the average DAR. Reductions in payload conjugated peak area and increases in unconjugated peak area, when compared to the T=0 sample, was used to assess the % of payload deconjugation.

[0578] Results

[0579] The payload deconjugation study (Figure 8) indicated that payload deconjugation was observed at low levels (< 10%) over 3 and 5 days. Payload deconjugation increased as a function of time with higher levels of payload loss after 5 days compared to after 3 days; the increase in deconjugated payload over time appears to be linear, not exponential. Payload loss was observed almost exclusively from the heavy chains with limited evidence of payload loss from the light chains (Table 7).

[0580] Of the 5 lead bispecific ADCs tested, BVX01-a0032-AB4A performed the best with the lowest percent of Bi-Fab deconjugation after 5 days (3.31 % payload loss), with BVX01-a0054-AB4A performing the worst with 9.73 % payload loss after 5 days (Table 7). BVX01-a0092-AB4A had the second lowest Bi-Fab deconjugation after 5 days (4.86 % payload loss) but suffered from other production and stability issues. As such, this data is supportive of choosing BVX01-a0032- AB4A and BVX01-a0059-AB4A as the top two lead bispecific ADCs.

[0581] Table 7 - The calculated average DAR of BVX01-a0032-AB4A, BVX01-a0054-AB4A, BVX01- a0059-AB4A and BVX01-a0092-AB4A after 0, 3 and 5 days at 40 °C. The average DAR of each chain (LC1 , LC2 and HCs) is calculated separately, then combined to give an overall DAR. The calculated % DAR loss for day 3 and 5 is relative to the corresponding DAR at Day 0.

[0582] Example 6 - Ex vivo safety studies

[0583] Ex vivo studies show good safety profile of the lead bispecific ADCs, where no toxicity to CD7+ T-cells or CD33+ myeloid progenitors / mature CD33+ cells is observed. Reagents

[0584] BVX01-a0032-AB4A (antiCD33 x antiCD7 Bi-Fab ADC) In House

[0585] BVX01-a0054-AB4A (antiCD33 x antiCD7 Bi-Fab ADC) In House

[0586] BVX01-a0059-AB4A (antiCD33 x antiCD7 Bi-Fab ADC) In House

[0587] BVX01-a0090-AB4A (antiCD33 x antiCD7 Bi-Fab ADC) In House BVX01-a0092-AB4A (antiCD33 x antiCD7 Bi-Fab ADC) In House

[0588] AntiCD7 x antiCD7 homodimer ADC In House

[0589] Human Cord Blood CD34+ Cells (mixed donor) (#70008.1) Stemcell Technologies

[0590] MethoCult™ Optimum Without EPO (#04437) Stemcell Technologies

[0591] Iscove's MDM with 2% FBS (#7700) Stemcell Technologies

[0592] SmartDish (#27370) Stemcell Technologies

[0593] STEMgrid™-6 (#27000) Stemcell Technologies

[0594] Hu PBMC (#70025.2) Stemcell Technologies

[0595] Dynabeads™ Untouched™ Human T Cells Kit (#11344D) Invitrogen

[0596] Dynabeads® Human T-Activator CD3 / CD28 (#11161 D) Gibco

[0597] Clear bottom 96-well plates CytoOne®, Non-Treated (#CC7672-7596) STARLABS

[0598] Disposable PS Reservoirs-StarTub PS (#E2310-1010) STARLABS

[0599] Cell Proliferation Reagent WST-1 (#ab155902) Abeam

[0600] 2mL Polypropylene Deep Well Plate (#AXYPDW20CS) SLS

[0601] RPMI-1640 medium (#21875059) Life Technologies

[0602] Fetal Bovine Serum, Heat inactivated (#11533387) Life Technologies

[0603] Methods

[0604] Methocult was thawed at 4°C overnight. Methocult was shaken and allowed to stand at room temperature until bubbles had dispersed and aliquoted into 3 ml aliquots. Concentrations of 0.1 nM, 1 nM and 10nM were prepared for each ADC in PBS. Each concentration was tested across duplicate wells. CD34+cells were thawed at 37°C and suspended in 2% FBS IMDM. 270 pl of suspension containing 3000 CD34+cells were pipetted into each 1 .5 ml tube containing 30 pl ADC and the solution was gently mixed by pipetting. The cell suspension / ADC mix was pipetted into a 3ml aliquot of Methocult, vortexed for 5 seconds and allowed to stand at room temperature until the bubbles had disappeared. Using an 18-gauge blunt needle and 6 ml syringe, 1 ml of cell / ADC / Methocult mix was transferred to a well of a Smart dish. Each plate was rocked to ensure the Methocult covered the entire well surface evenly and then placed in a 37°C, 5% CO2 incubator. The colonies were counted on day 10 and the data plotted using PRISM software.

[0605] Healthy T cells were isolated from huPBMCs by using a Dynabeads™ Untouched™ Human T Cells Kit, following the manufactures protocol. After isolation, T cells were counted and activated by resuspending 1x106cells / ml into 10 ml of RPMI-1640 media containing 10% FBS, 20 ng / ml interleukin-2 (IL-2) and a 1-1 ratio of CD3 / CD28 beads. Following activation, T cells were dispensed at 50,000 cells per well into 96 well plates in a total volume of 50 pl. 9 point 3-fold ADC titrations were prepared in RPMI-1640 10% FBS to a final dose range of 30 nM-0.0152 nM, by using a deep-well plate. 50 pl of drug dilution was added to the 96 well plates containing the activated T cells to a final volume of 100 pl. Plates were incubated at 37°C, 5% CO2 for 96 h. After incubation, 10 pl of WST-1 Assay Reagent was added to each well and plates were incubated at 37°C, 5% CO2 for 3h. Absorbance was measured at 440 nm and 620 nm on a Spectramax M2 (Molecular Devices) and dose-curve plots were obtained by using PRISM software.

[0606] Results

[0607] Figures 9 and 10 show that the lead bispecific ADCs do not cause any decrease in colony formation of CD33+ healthy human myeloid cells (derived from healthy human cord blood donors) in a CFU assay or cause any toxicity to activated CD7+ T-cells within a cell kill assay, where T-cells have been isolated from healthy huPBMCs.

[0608] Example 7 - Assessment of Potential Off-Target Toxicity of Lead bispecific ADCs via Macropinocytosis

[0609] Reagents

[0610] HUVEC cells Thermo Fisher

[0611] BVX01-a0032-AB4A (antiCD33 x antiCD7 Bi-Fab ADC) In house

[0612] BVX01-a0059-AB4A (antiCD33 x antiCD7 Bi-Fab ADC) In house

[0613] Clear bottom 96-well plates CytoOne®, Non-Treated (#CC7672-7596) STARLABS Disposable PS Reservoirs-StarTub PS (#E2310-1010) STARLABS

[0614] Cell Proliferation Reagent WST-1 (#ab155902) Abeam

[0615] 2mL Polypropylene Deep Well Plate (#AXYPDW20CS) SLS

[0616] Human Large Vessel Endothelial Cell Basal Media (#M200500) Thermo Fisher Low Serum Growth Supplement (S00310) Thermo Fisher

[0617] Fetal Bovine Serum, Heat inactivated (#11533387) Life Technologies

[0618] Methods

[0619] Fabs were modified to permit Bi-Fab formation by bioorthogonal reactive partners. On formation of the Bi-Fab, it was conjugated with mcMMAF average DAR 4. HUVEC cell lines (known to have the ab...

Claims

CLAIMS1 . A bispecific binding molecule, comprising a first antigen binding domain that binds to CD7 and a second antigen binding domain that binds CD33, wherein said first antigen binding domain comprises ai) a heavy chain variable (VH) region comprising SEQ ID NO. 21 or a sequence comprising at least 90%, 92%, 95%, 97% or 99% sequence identity thereto, and aii) a light chain variable (VL) region comprising SEQ ID NO. 23 or a sequence comprising at least 80%, 85%, 90%, 92%, 95%, 97% or 99% sequence identity thereto, or bi) a heavy chain variable (VH) region comprising SEQ ID NO. 25 or a sequence comprising at least 90%, 92%, 95%, 97% or 99% sequence identity thereto, and bii) a light chain variable (VL) region comprising SEQ ID NO. 27 or a sequence comprising at least 80%, 85%, 90%, 92%, 95%, 97% or 99% sequence identity thereto, or ci) a heavy chain variable (VH) region comprising SEQ ID NO. 29 or a sequence comprising at least 90%, 92%, 95%, 97% or 99% sequence identity thereto, and cii) a light chain variable (VL) region comprising SEQ ID NO. 31 or a sequence comprising at least 80%, 85%, 90%, 92%, 95%, 97% or 99% sequence identity thereto, or di) a heavy chain variable (VH) region comprising SEQ ID NO. 33 or a sequence comprising at least 90%, 92%, 95%, 97% or 99% sequence identity thereto, and dii) a light chain variable (VL) region comprising SEQ ID NO. 35 or a sequence comprising at least 80%, 85%, 90%, 92%, 95%, 97% or 99% sequence identity thereto, or ei) a heavy chain variable (VH) region comprising SEQ ID NO. 37 or a sequence comprising at least 90%, 92%, 95%, 97% or 99% sequence identity thereto, and eii) a light chain variable (VL) region comprising SEQ ID NO. 39 or a sequence comprising at least 80%, 85%, 90%, 92%, 95%, 97% or 99% sequence identity thereto.

2. The bispecific binding molecule of claim 1 , wherein the bispecific antibody binding molecule is capable of targeting cells expressing CD7+CD33+, and wherein CD33 is expressed either at a high or a low level.

3. The bispecific binding molecule of claims 1 or 2, wherein the first antigen binding domain comprises ai) a heavy chain variable (VH) region comprising SEQ ID NO. 21 or a sequence comprising between 1 and 19 amino acid differences thereto, and aii) a light chain variable (VL) region comprising SEQ ID NO. 23 or a sequence comprising between 1 and 21 amino acid differences thereto, or bi) a heavy chain variable (VH) region comprising SEQ ID NO. 25 or a sequence comprising between 1 and 19 amino acid differences thereto, and bii) a light chain variable (VL) region comprising SEQ ID NO. 27 or a sequence comprising between 1 and 21 amino acid differences thereto, or ci) a heavy chain variable (VH) region comprising SEQ ID NO. 29 or a sequence comprising between 1 and 19 amino acid differences thereto, andcii) a light chain variable (VL) region comprising SEQ ID NO. 31 or a sequence comprising between 1 and 21 amino acid differences thereto, or di) a heavy chain variable (VH) region comprising SEQ ID NO. 33 or a sequence comprising between 1 and 19 amino acid differences thereto, and dii) a light chain variable (VL) region comprising SEQ ID NO. 35 or a sequence comprising between 1 and 21 amino acid differences thereto, or ei) a heavy chain variable (VH) region comprising SEQ ID NO. 37 or a sequence comprising between 1 and 19 amino acid differences thereto, and eii) a light chain variable (VL) region comprising SEQ ID NO. 39 or a sequence comprising between 1 and 21 amino acid differences thereto.

4. The bispecific binding molecule according to any preceding claim, wherein the first antigen binding domain comprises i) a heavy chain variable (VH) region comprising a sequence selected from SEQ ID NO.21 , SEQ ID NO. 25, SEQ ID NO. 29, SEQ ID NO. 33, or SEQ ID NO. 37 and ii) a light chain variable (VL) region comprising a sequence selected from SEQ ID NO. 23, SEQ ID NO. 27, SEQ ID NO. 31 , SEQ ID NO. 35, or SEQ ID NO. 39.

5. The bispecific binding molecule according to any preceding claim wherein the first antigen binding domain comprises i) a heavy chain comprising SEQ ID NO. 22 or a sequence comprising at least 70%, 80%, 85%, 90%, 95% or 99% sequence identity thereto, and ii) a light chain comprising SEQ ID NO. 24 or a sequence comprising at least 70%, 80%, 85%, 90%, 95% or 99% sequence identity thereto.

6. The bispecific binding molecule according to any preceding claim, wherein the first antigen binding domain comprises i) a heavy chain comprising a sequence selected from SEQ ID NO. 22, SEQ ID NO. 26, SEQ ID NO. 30, SEQ ID NO. 34, or SEQ ID NO. 38 and ii) a light chain variable (VL) region comprising a sequence selected from SEQ ID NO. 24, SEQ ID NO. 28, SEQ ID NO. 32, SEQ ID NO. 36, or SEQ ID NO. 40.

7. The bispecific binding molecule according to any preceding claim, wherein the second antigen binding domain comprises i) a heavy chain variable (VH) region comprising a HC CDR1 comprising SEQ ID NO. 2, a HC CDR2 comprising SEQ ID NO. 3, a HC CDR3 comprising SEQ ID NO. 4, and ii) a light chain variable (VL) region comprising a LC CDR1 comprising SEQ ID NO. 7, a LC CDR2 comprising SEQ ID NO. 8, a LC CDR3 comprising SEQ ID NO. 9.

8. The bispecific binding molecule according to any preceding claim, wherein the second antigen binding domain comprises i) a heavy chain variable (VH) region comprising SEQ ID NO. 1 and ii) a light chain variable (VL) region comprising SEQ ID NO. 6.

9. The bispecific binding molecule of any preceding claim, wherein the first antigen binding domain has a binding affinity (KD) to CD7 of between 5 x1 O-10M and 2 x10-7M,10. The bispecific binding molecule of any preceding claim, wherein the second antigen binding domain has a binding affinity (KD) to CD33 of between 1 .5 x10-10M and 3 x10-8M.

11. The bispecific binding molecule of any preceding claim, wherein said antibody or antigen binding fragment is capable of inducing CD33 and / or CD7 receptor mediated internalization into a CD33+ and / or CD7+ cell.

12. The bispecific binding molecule of any preceding claim, wherein said antibody or antigen binding fragment is capable of selectively targeting cells expressing CD7+ and CD33+, compared to cells expressing CD7+ or cells expressing CD33+.

13. The bispecific binding molecule of any preceding claim, wherein said first or second antigen binding domain comprises a Fab, Fab’, F(ab)2, scFv, (scFv), heavy chain only antibody, single domain antibody, nanobody.

14. The bispecific binding molecule of any preceding claim, wherein the first and second antigen binding domain are attached via a linker.

15. The bispecific binding molecule of any preceding claim, wherein the first and second antigen binding domain are conjugated together via click chemistry.

16. The bispecific binding molecule of claim 15, wherein the click chemistry used to conjugate together the first and second antigen binding domain is selected from a thiol-ene reaction, a copper catalyzed azide-alkyne cycloaddition (CuAAC) reaction, a strained-promoted azidealkyne click chemistry (SPAAC) reaction and / or an inverse electron demand Diels-Alder (iEDDA) reaction, for example via an inverse electron demand Diels-Alder (iEDDA) reaction.

17. The bispecific binding molecule of any preceding aspect, wherein said antibody or antigen binding fragment is conjugated to a payload.

18. The bispecific binding molecule of any preceding claim, wherein the payload is a cell killing agent, an immune-modulating payload, a macrophage class switching agent or a light activatable payload.

19. The bispecific binding molecule of any preceding claim, wherein the immune-modulating payload is a STING agonist or a toll-like receptor agonist.

20. The bispecific binding molecule of claim 18, wherein the cell killing agent comprises a cytotoxin.21 . The bispecific binding molecule of claim 20, wherein said cytotoxin is selected from: i) a peptide toxin; or ii) a chemical toxin.

22. The bispecific binding molecule of any one of claims 17 to 21 , wherein the first antigen binding domain has a binding affinity (KD) to CD7 of between 6.5 x10-1° M and 2 x10-7M.

23. The bispecific binding molecule of any preceding claim, further comprising a linker for linking the payload to the bispecific antibody or antigen binding fragment.

24. The bispecific binding molecule of claim 23, wherein the linker is attached to the bispecific antibody or antigen binding fragment via a non-naturally occurring amino acid residue present in the bispecific antibody or antigen binding fragment.

25. The bispecific binding molecule of claim 24, wherein the non-naturally occurring amino acid residue is selected from a cysteine residue, a lysine residue, a histidine residue, a tyrosine residue, formylglycine residue.

26. The bispecific binding molecule of any one of claims 30 to 32, wherein the linker is selected from one or more of a cleavable linker, a non-cleavable linker, a pH sensitive linker, a redox sensitive linker.

27. The bispecific binding molecule of any preceding claim, further comprising an additional moiety selected from a half-life extending moiety and / or a label.

28. The bispecific binding molecule of claim 27, wherein the half-life extending moiety is selected from serum albumin protein, a binding molecule that binds serum albumin, an Fc domain and / or a modified Fc domain.

29. The bispecific binding molecule of any preceding claim, further comprising one or more additional binding domains.

30. An isolated nucleic acid molecule encoding the bispecific antibody or antigen binding fragment of any one of claims 1 to 29.31 . A vector encoding the isolated nucleic acid of claim 30.

32. A host cell comprising the nucleic acid of claim 30, or the vector of claim 31 .

33. An immunoconjugate comprising the bispecific binding molecule of any of claims 1 to 29.

34. A pharmaceutical composition comprising the bispecific binding molecule of any one of claims 1 to 29 and optionally a pharmaceutically acceptable excipient, and / or adjuvant.

35. The bispecific binding molecule according to any one of claims 1 to 29 for use in the treatment of a malignancy.

36. A method of treating or preventing a malignancy comprising administering a therapeutically effective amount of the bispecific antibody or antigen binding fragment of any one of claims 1 to 29.

37. The use of the bispecific binding molecule of any one of claims 1 to 29 for the manufacture of a medicament for the treatment of a malignancy.

38. The bispecific binding molecule for use according to claim 35, the method according to claim 36 of the use according to claim 37, wherein the malignancy is selected from one of the following cancers: haematological cancers or Acute Myeloid Leukaemia.

39. The bispecific binding molecule for use according to claim 40, the method according to claim 36 of the use according to claim 37, wherein the malignancy is an Acute Myeloid Leukaemia (AML)or AML derived cancer.

40. An in vitro, ex vivo or in vivo method of detecting CD33+ / CD7+ cells within a biological sample, comprising obtaining a biological samplecontacting said biological sample with a bispecific binding molecule according to any one of claims 1 to 29.41 . An in vitro, ex vivo or in vivo method of delivering a payload to CD33+ / CD7+ cells within a biological sample, comprising obtaining or providing a biological sample contacting said biological sample with a bispecific binding molecule according to any one of claims 1 to 29.

42. A method for reducing off target toxicity of a cancer treatment comprising administering to a subject a bispecific binding molecule according to any one of claims 1 to 29.

43. A kit comprising the bispecific binding molecule of any one of claims 1 to 29 and optionally instructions for use.

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