Therapeutic agents targeting CLPTM1

Abstract

A therapeutic agent capable of binding to the receptor CLPTM1 at the surface of an immune cell and modulating its activity for use in modulating the activity of the immune system to treat cancer, wherein the therapeutic agent is capable of inhibiting the growth and/or viability of an anti-inflammatory and/or immunosuppressive cell to relieve unwanted or deleterious immunosuppression by eliminating anti-inflammatory and/or immunosuppressive immune cells; and/or the therapeutic agent is capable of stimulating an antigen-presenting immune cell and acts to stimulate antigen-presenting immune cells to activate an anti-cancer immune response.

Claims

1. A product comprising: (a) a therapeutic agent capable of binding to CLPTM1 at the surface of a cell and inhibiting the growth and/or viability of said cell; and (b) an immune checkpoint inhibitor, the immune checkpoint inhibitor being an antibody which binds to an immune checkpoint protein or to a receptor for an immune checkpoint protein; wherein said therapeutic agent and said immune checkpoint inhibitor are formulated for combined administration separately, sequentially or simultaneously, and wherein the therapeutic agent is selected from: (i) an antibody having immune effector function selected from antibody dependent cell-mediated cytotoxicity (ADCC), antibody dependent cell-mediated phagocytosis (ADCP), and/or complement dependent cytotoxicity (CDC); and (ii) a conjugate comprising an antibody conjugated to a cytotoxic or cytostatic drug, wherein said antibody is capable of being internalized by the cell, wherein said antibody in said therapeutic agent is capable of binding to CLPTM1 and comprises the complementarity-determining regions (CDRs) VLCDR1, VLCDR2, VLCDR3, VHCDR1, VHCDR2 and VHCDR3, and wherein (i) each of said CDRs has an amino acid sequence as follows: VLCDR1 has the sequence set forth in SEQ ID NO: 66; VLCDR2 has the sequence set forth in SEQ ID NO: 67; VLCDR3 has the sequence set forth in SEQ ID NO: 68; VHCDR1 has the sequence set forth in SEQ ID NO: 69; VHCDR2 has the sequence set forth in SEQ ID NO: 70; and VHCDR3 has the sequence set forth in SEQ ID NO: 71; or (ii) each of said CDRs has an amino acid sequence as follows: VLCDR1 has the sequence set forth in SEQ ID NO: 72; VLCDR2 has the sequence set forth in SEQ ID NO: 73; VLCDR3 has the sequence set forth in SEQ ID NO: 74; VHCDR1 has the sequence set forth in SEQ ID NO: 75; VHCDR2 has the sequence set forth in SEQ ID NO: 76; and VHCDR3 has the sequence set forth in SEQ ID NO: 77.

2. The product of claim 1, wherein the therapeutic agent is capable of inhibiting the growth and/or viability of an anti-inflammatory and/or immunosuppressive cell to relieve unwanted or deleterious immunosuppression by eliminating anti-inflammatory and/or immunosuppressive immune cells; and/or wherein the immune checkpoint inhibitor is an anti-PD1, anti-PDL1 or an anti-CTLA-4 antibody.

3. The product of claim 1 wherein the therapeutic agent is a conjugate comprising the antibody conjugated to a tubulin inhibitor.

4. A therapeutic agent capable of binding to CLPTM1 at the surface of an immune cell and modulating its activity, wherein (i) the therapeutic agent is capable of inhibiting the growth and/or viability of an anti-inflammatory and/or immunosuppressive cell; and/or (ii) the therapeutic agent is capable of stimulating an antigen-presenting immune cell; wherein each said therapeutic agent (i) and (ii) is a conjugate comprising an antibody capable of binding to CLPTM1, conjugated to a drug; and wherein said antibody comprises the complementarity-determining regions (CDRs) VLCDR1, VLCDR2, VLCDR3, VHCDR1, VHCDR2 and VHCDR3, wherein (i) each of said CDRs has an amino acid sequence as follows: VLCDR1 has the sequence set forth in SEQ ID NO: 66; VLCDR2 has the sequence set forth in SEQ ID NO: 67; VLCDR3 has the sequence set forth in SEQ ID NO: 68; VHCDR1 has the sequence set forth in SEQ ID NO: 69; VHCDR2 has the sequence set forth in SEQ ID NO: 70; and VHCDR3 has the sequence set forth in SEQ ID NO: 71; or (ii) each of said CDRs has an amino acid sequence as follows: VLCDR1 has the sequence set forth in SEQ ID NO: 72; VLCDR2 has the sequence set forth in SEQ ID NO: 73; VLCDR3 has the sequence set forth in SEQ ID NO: 74; VHCDR1 has the sequence set forth in SEQ ID NO: 75; VHCDR2 has the sequence set forth in SEQ ID NO: 76; and VHCDR3 has the sequence set forth in SEQ ID NO: 77.

5. The product of claim 1, wherein said antibody in said therapeutic agent is: (i) a half-molecule antibody fragment; (ii) a single-chain antibody; (iii) an afucosylated monoclonal antibody; (iv) a humanised or chimeric antibody; or (v) a human antibody.

6. The product of claim 1, wherein in said conjugate of part (ii), said drug is a radioisotope or a toxin or a small molecule compound or a chemotherapy drug.

7. The product of claim 6, wherein said drug is (i) a maytansinoid, or (ii) mertansine, emtansine or monomethyl auristatin E(MMAE).

8. The product of claim 1, wherein said therapeutic agent is capable of inhibiting the growth and/or viability of an anti-inflammatory and/or immunosuppressive cell selected from or including: (i) immunosuppressive immune cells; (ii) tumour-infiltrating lymphocytes; (iii) Treg or macrophage cells; or (iv) M2 macrophage cells.

9. The product of claim 1, wherein said checkpoint inhibitor is an antibody against PDL-1, PD-1, CTLA4, TIM3, CD137, CD223, or phosphatidylserine.

10. A method of treating melanoma or breast cancer in a subject, or reducing cancer metastasis to the lung in a subject, said method comprising administering to said subject a therapeutically effective amount of the product of claim 1.

11. The method of claim 10, wherein said therapeutic agent is the antibody having immune effector function.

12. The method of claim 10, wherein the antibody is IgG.

13. The method of claim 12, wherein said IgG is IgG1 or IgG3.

14. The method of claim 10, wherein said therapeutic agent is the conjugate comprising an antibody capable of binding to CLPTM1 conjugated to a cytotoxic or cytostatic drug, optionally wherein the antibody is conjugated to the drug by a linker.

15. The method of claim 10, wherein said antibody is: (i) a half-molecule antibody fragment; (ii) a single-chain antibody; (iii) an afucosylated monoclonal antibody; (iv) a humanised or chimeric antibody; or (v) a human antibody.

16. The method of claim 14, wherein said drug is a radioisotope or a toxin or a small molecule compound or a chemotherapy drug.

17. The method of claim 16, wherein said drug is (i) a tubulin inhibitor; (ii) a maytansinoid, or (iii) mertansine, emtansine or monomethyl auristatin E (MMAE).

18. The method of claim 10, wherein (a) said therapeutic agent is capable of inhibiting the growth and/or viability of an anti-inflammatory and/or immunosuppressive cell selected from or including: (i) immunosuppressive immune cells; (ii) tumour-infiltrating lymphocytes; (iii) Treg or macrophage cells; or (iv) M2 macrophage cells.

19. The method of claim 18, wherein the immune checkpoint inhibitor is an anti-PD1, anti-PDL1 or an anti-CTLA-4 antibody.

20. A product comprising: (a) a therapeutic agent capable of binding to CLPTM1 at the surface of antigen-presenting cells (APC) selected from dendritic cells and macrophage cells and stimulating said APC to activate an immune response; and (b) an immune checkpoint inhibitor, being an antibody which binds to an immune checkpoint protein, or to a receptor for an immune checkpoint protein; wherein said therapeutic agent and said immune checkpoint inhibitor are formulated for combined administration separately, sequentially or simultaneously; and wherein the therapeutic agent is an antibody-drug conjugate (ADC) wherein the drug comprises a tubulin inhibitor and the antibody is capable of binding specifically to CLPTM1 and comprises the complementarity-determining regions (CDRs) VLCDR1, VLCDR2, VLCDR3, VHCDR1, VHCDR2 and VHCDR3, wherein (i) each of said CDRs has an amino acid sequence as follows: VLCDR1 has the sequence set forth in SEQ ID NO: 66; VLCDR2 has the sequence set forth in SEQ ID NO: 67; VLCDR3 has the sequence set forth in SEQ ID NO: 68; VHCDR1 has the sequence set forth in SEQ ID NO: 69; VHCDR2 has the sequence set forth in SEQ ID NO: 70; and VHCDR3 has the sequence set forth in SEQ ID NO: 71; or (ii) each of said CDRs has an amino acid sequence as follows: VLCDR1 has the sequence set forth in SEQ ID NO: 72; VLCDR2 has the sequence set forth in SEQ ID NO: 73; VLCDR3 has the sequence set forth in SEQ ID NO: 74; VHCDR1 has the sequence set forth in SEQ ID NO: 75; VHCDR2 has the sequence set forth in SEQ ID NO: 76; and VHCDR3 has the sequence set forth in SEQ ID NO: 77.

21. A method of treating melanoma or breast cancer in a subject, or reducing cancer metastasis to the lung in a subject, said method comprising administering to said subject a therapeutically effective amount of the product of claim 20.

22. A method of modulating the immune system within a tumor microenvironment in a subject, said method comprising administering to the subject a therapeutically effective amount of the product of claim 1.

23. A method of modulating the immune system within a tumor microenvironment in a subject, said method comprising administering to the subject a therapeutically effective amount of the product of claim 20.

24. The product of claim 20, wherein said tubulin inhibitor is (i) a maytansinoid, or (ii) mertansine, emtansine or monomethyl auristatin E.

Description

(1) The present invention may be better understood through the following Examples and Figures, in which:

(2) FIG. 1 shows that CD14+ immune cells express CLPTM1 at their surface. CD14+ monocytes from peripheral blood were stained for CLPTM1. Dotted line—negative FMO control. Middle peak—Extra-cellular CLPTM1 staining. Right hand peak—Total (extra-cellular & intracellular CLPTM1 staining).

(3) FIG. 2 shows that CLPTM1 is at the surface of CD14+ macrophages with low levels of MHC2.

(4) FIG. 3 shows that T-cells with low levels of TMEM173 expression express CLPTM1 at their cell surface. CD45+; CD19-; CD3+ tumour infiltrating lymphocytes were analysed.

(5) FIG. 4 shows that M2 macrophages, but not dendritic cells or M1 macrophages, express high levels of CLPTM1.

(6) FIG. 5 shows that mouse IgG2a-type antibodies are capable of eliciting strong ADCP.

(7) FIG. 6 shows that an antibody conjugated to a cytotoxic drug are effective at eliminating cells expressing CLPTM1 at their cell surface.

(8) FIG. 7 shows combined results of a peptide screen to identify residues within the extracellular domain of CLPTM1 that are required for binding to GDF15 measuring binding on a peptide array. The results of binding of GDF15 to immobilised peptides are shown.

(9) FIG. 8 shows the expression of CLPTM1 at the cell surface for various cells of the immune system and in tumour cells isolated from the following mouse cancer model: 4T1 (breast), B16 (melanoma), CT26 (colon) and LLC (lung), and from healthy spleen tissue. High levels of expression are observed in various cells of the immune system isolated from the different tumour microenvironments.

(10) FIG. 9 shows the effect of an anti-CLPTM1 antibody (7G12) conjugated to DM1 (ADC) on tumour growth in a B16 mouse melanoma model. A) Tumour weight (g) at end of study. B) Tumour size (mm.sup.3) over time. The ADC reduces tumour growth to a similar extent to an anti-PD1 antibody (aPD1), compared to isotype controls and the 7G12 antibody alone, and the combined therapy of the ADC and aPD1 results in a greater reduction in tumour growth than either treatment in isolation.

(11) FIG. 10 shows the expression of PDL1 at the cell surface in pDC cells isolated from tumour infiltrating lymphocytes (TILs). PDL1 expression is a marker of immune activation. The anti-CLPTM1 antibody (7G12) conjugated to DM1 (ACD) induces a higher level of PDL1 expression than either an isotype control or the 7G12 antibody alone. The combination of the ADC and an anti-PD1 antibody causes a significant increase in PDL1 expression at the cell surface relative to the isotype control and 7G12 antibody alone.

(12) FIG. 11 shows the induction of cytokine expression, and the correlation between levels of cytokines and tumour size in serum isolated from the mice in the Example 6 study. A), C), E) and G) levels of CCL2. CCL3. CCL5 and CXCL9 in response to different treatments. The ADC and aPD1 combination were found to increase all of the tested cytokines above the control level. B). D). F) and H) levels of CCL2, CCL3, CCL5 and CXCL9 correlated to tumour size (g). Higher levels of the cytokines are linked to smaller tumour size. I), J) levels of CCL5 in response to ADC and aPD1 combination treatment and ADC only. Higher levels of the cytokine are linked to small tumour size. Together, these data demonstrate immune activation against cancers.

(13) FIG. 12 shows the effect of an anti-CLPTM1 antibody (59D04) conjugated to DM1 (ADC) on tumour growth in a B16 mouse melanoma model. The ADC and aPD1 combination treatment resulted in reduced tumour growth relative to the ADC or aPD1 alone, and relative to an isotype control.

(14) FIG. 13 shows the expression of CLPTM1 at the cell surface in M2 macrophages and activation of macrophages by the 59D04 antibody conjugated to DM1 (ADC). A) Cell surface expression of CLPTM1 measured by flow cytometry. B), C) expression of CD80 and CD86 on the cell surface in a dilution series of the ADC. Both CD80 and CD86 were stimulated by the ACD, relative to the 59D04 antibody alone or an isotype control.

(15) FIG. 14 shows internalisation of the 59D04 antibody conjugated to phRhodo. Both IgG1 and IgG2a isotype antibodies show a high level of internalisation. Isotype control and the “Santa Cruz” anti-CLPTM1 antibody show very low levels of internalisation.

(16) FIG. 15 shows the measurement of the affinity of the 59D04, 7G12, Santa Cruz and AbCam antibodies by flow cytometry. CLPTM1 binding in permeabilised 0-876 cells expressing native CLPTM1 was measured in a 10× dilution series. The highest affinity antibody measured was 7G12, which retained greater than 50% of its maximum binding at 0.1 μg/ml. The AbCam antibody also retained greater than 50% of its maximum binding at 0.1 μg/ml. The 59D04 antibody retained greater than 50% of its maximum binding at 1 μg/ml, whilst the Santa Cruz antibody had less than 50% of its maximum binding at this level.

(17) FIG. 16 shows the effect of an anti-CLPTM1 antibody (59D04) as an IgG2A antibody or as a conjugate with DM1 (59D04-DM1) alone or in combination with an anti-PD1 antibody (aPD1), compared to aPD1 or an isotype control on the syngeneic orthotopic breast cancer model 4T1 in mouse. A) Primary tumour weight (g) at end of study. B) Number of lung metastases. Whilst there was no statistically significant change in primary tumour size, the number of metastases was affected both by the 59D04 IgG2A antibody and the 59D04-DM1 conjugate, compared to isotype antibody control.

EXAMPLES

Example 1—Expression of CLPTM1 on Immune Cells

(18) Materials and Methods

(19) Cell Isolation, Selection and Culture

(20) The cell suspension of heparinized peripheral blood was diluted 1:1 and placed on a Ficoll Paque Plus, after which the suspension was centrifuged at 400×g for 30 minutes. The PMBC layer was transferred to 50 mL tube of PBS and centrifuged for 7 min at 250×G. CD14+ cells were isolated using EasySep Positive Selection kit CD14 (Stemcell inc) according to the manufacturer's instructions. Cell culture was performed in 48 well cell culture plates which were seeded with cell suspension at a 0.833×10.sup.6 cells/mL in RPMI medium and 300 uL added to each well. CD14+ cells were allowed to adhere for 90 minutes at 37° C. in an incubator and non-adherent cells were removed.

(21) Derivation of M1, M2 and Dendritic Cells (DC) from CD14+ Monocytes

(22) PBMCs from buffy coat of healthy blood donor were obtained and CD14 positive cells were isolated and plated in 0.5 ml 1640 RPMI, 1×10{circumflex over ( )}6 cells/ml. For M2 macrophage derivation, 50 ng/ml M-CSF was added. For M1 derivation 50 ng/ml GM-CSF was added. For DC derivation 50 ng/ml GM-CSF+20 ng/ml IL-4 was added, and 3 days before harvest 20 ng/ml TNFα. All cells were harvested after 7 day total culture. Medium and cytokines were changed every 2-3 days. M1 and M2 differentiation cultures were in a separate reaction also given 20 μg/ml of the 7G12 anti-CLPTM1 antibody 24 hours before harvest and flow cytometry analysis. All cytokines purchased from RnD Systems.

(23) Flow Cytometry

(24) The amount of surface expression of CLPTM1 was analyzed by flow cytometry using the 3A10-PE antibody. 3A10 mouse monoclonal antibody was conjugated to phycoerythrin using Lightning link kit from Innova Biosciences. FIG. 4 shows that CLPTM1 surface expression more than 100× higher on M2 macrophages illustrating the applicability of this receptor as a suitable drug target for cell elimination by various means in cancer. Addition of the 7G12 antibody further increased the CLPTM1 surface expression but only on the M2 macrophages and did not increase CLPTM1 on the surface of M1 macrophages.

Example 2—ADCP Assay

(25) Materials and Methods

(26) Mouse monoclonal antibodies were developed by immunization with synthetic peptides using Rapid-Prime™ method by ImmunoPrecise (Victoria, Canada). The following peptide sequences where used giving rise to corresponding antibody clones: PWNFLGDELYEQSDE (SEQ ID NO: 48) (3A10, 7G12), DEEQDSVKVALLET (SEQ ID NO: 49) (2D12), TEADPEMIKRAEDY-C(SEQ ID NO: 51) (5H8, 10F4, 6E4), C-GDYYPIIYFNDYW (SEQ ID NO: 53) (1G10, 3G11, 10F3, 6A1), C-RNLFPKDTLMNLH (SEQ ID NO: 55) (9E3). The “-C” or “C-” in SEQ ID NOs: 51, 53 and 55 indicates that a cysteine (C) residue has been added to the native sequence for conjugation purposes, and is not part of the natural amino acid sequence. (The corresponding native sequences are shown in SEQ ID NOs: 50, 52 and 54 respectively).

(27) J774 cells were stained with either CellTrace Violet (ThermoFisher C34557) or CellTrace Green (ThermoFisher C34554) according to the manufacturer's instructions. Cell cultures containing 50% of each labelled cell where grown overnight with 20 ug/ml of each monoclonal antibody. Double positive cells were detected and scored as a percentage of total cell population using flow cytometry FACSCANTOII (Becton Dickinson).

(28) Mouse macrophage cell line J774 with high cell surface expression of CLPTM1 was cultured. These cells where labeled with a red or violet fluorescent dye and co-cultured overnight with various CLPTM1 antibodies.

(29) Antibody Dependent Cellular Phagocytosis (ADCP) was assessed for several mouse monoclonal antibodies binding to CLPTM1. These antibodies bind to different epitopes on CLPTM1 and have various effector functions on their Fc. Mouse macrophage cell line J774 was used due to its high expression of surface CLPTM1 as we found by flow cytometry analysis. These cells were labelled with a red or violet fluorescent dye and co-cultured overnight with various CLPTM1 antibodies. Flow cytometry analysis revealed the % of double labeled cells showing the level of phagocytosis. Only IgG2a effector functionalized mAb clones are capable of eliciting strong ADCP. Our data in FIG. 5 show that clone 5H8 and 1G10, both IgG2a-type, increase phagocytosis.

(30) These data indicate that antibodies having immune effector function can lead to the elimination of cells expressing CLPTM1.

Example 3—Effect of Cytotoxic Drug Conjugate on Cells Expressing CLPTM1

(31) Materials and Methods

(32) CLPTM1 clone 7G12 (IgG1) was conjugated to Mertansine DM1 (Abcam ab146096) using an SMCC crosslinker (ThermoFisher 22360). 100 ug of antibody in 100 uL was first PBS equilibrated in a Zeba spin column (ThermoFisher 89808). Then 10 uL of SMCC in DMSO (1.76 ug/uL) was added and set at room temperature for 2 hours. Surplus SMCC was removed by a Zeba spin column then mixed with 5 uLs of Mertansine (1.3 mM dissolved in dimethylformamide). Reaction was set overnight at room temperature then excess mertansine removed by Zeba spin column 7kMW-cut off.

(33) Cultures of 200,000 seeded mouse macrophage cells J774 with verified high levels of CLPTM1 expression were prepared. A control was prepared, in which no antibody was added to the culture. A negative control sample in which the unconjugated 7G12 antibody was used. 7G12 antibody conjugated to Mertansine was added to a test sample. Cells were seeded on Day 0, and grown for three days. After three days, manual counting was performed to establish cell numbers. The number of cells present in each condition is shown in FIG. 6.

(34) After three days' culture, the test sample to which Mertansine-conjugated antibody had been added demonstrated a large decrease in the number of cells present. By contrast, both the control and the negative control samples showed high levels of cell growth following three days' culture.

(35) This demonstrates that cytotoxic drug conjugates to antibodies are effective at eliminating cells expressing CLPTM1 at their cell surface.

Example 4—Epitope Mapping of ‘Bioss’ pAb on CLPTM1

(36) Materials and Methods

(37) Peptide Array

(38) The N-terminal aa 1-354 aa of CLPTM1 was divided with complete coverage into 86 unique 15-mers with 11 aa overlap, produced by JPT peptide Technologies GmbH. The Bioss 8018R polyclonal antibody was incubated at 1 μg/ml o/n at +4 C, washed extensively in PBST followed by 1 hour incubation at room temperature with Alexa flour 647 (Thermo scientific) Rabbit IgG (H+L) Polyclonal Secondary Antibody (Catalog #: A-21244) diluted 1:60000 and after repeated PBST wash detected using a G2502 Microarray scanner (Agilent Technologies). The polypeptides used are shown in FIG. 7.

(39) The epitope 1 for Bioss BS8018R has the highest Median fluorescence intensity (MFI) (signal intensity) on array, demonstrating highest binding affinity for this site.

(40) Binding of the antibody to the various peptides in the array is shown in FIG. 7A.

(41) Minimal common denominator: PKD (SEQ ID NO: 8)

(42) Most likely flanking amino acids also participate within the region: GGAPRVASRNLFPKDTLMNLHVYISEH (SEQ ID NO: 56).

(43) The epitope 2 displayed a slightly lower MFI, yet suggest a second site with affinity for this antibody. Binding of the antibody to the various peptides in the array is shown in FIG. 7B. The region for epitope 2 is within: LDQYVKFDAVSGDYYPIIYFNDYWNLQKDYYPINE (SEQ ID NO: 57).

Example 5—CLPTM1 Expression Levels on Various Immune Cells and Cancer Cells

(44) Four syngeneic mouse models of cancer were analyzed as of tumor infiltrating leucocytes and expression of CLPTM1. Data is illustrated in FIG. 8 as percentage of positive cells over isotype control and Mean Fluorescence Intensity (MFI) in comparison to immune cells isolated from healthy mouse spleen. The data show expression of CLPTM1 on various immune cells and the tumor cells themselves but no expression on immune cells from healthy spleen. Data was acquired by standard flow cytometry protocol using antibodies from Biolegend and Beckton-Dickinson on a FACS Fortessa instrument.

(45) Especially antigen presenting cells such as dendritic cells and macrophages show high surface expression of CLPTM1 as well as myeloid derived suppressor cells (MDSCs).

Example 6—B16-F10 Tumor Growth Data 7G12-DM1

(46) Antigen-Drug Conjugate (ADC) Preparation

(47) DM1-antibody conjugates were prepared as previously described in Example 3 but using a PEG4 linker in the SMCC (Pierce, ThermoFisher) in order to improve water solubility of the conjugate and scaled up to 20 mg of antibody, either 7G12 or a mouse IgG1 isotype control (BioXcell). A drug antibody ratio of 7.3 was achieved with for the 7G12-DM1 conjugate as analyzed by spectrophotometry at OD 252 and OD 280 using extinction coefficients 28044 and 5700 for DM1 at A252 and A280 respectively (M{circumflex over ( )}-1 cm{circumflex over ( )}-1) and 87360 and 224000 at A252 and A280 respectively for the antibody (NanoDrop). The isotype control showed DAR of 1.8. ADCs were stored at +4C in sterile conditions.

(48) In Vivo Models

(49) An in vivo effect study of the 7G12-DM1 ADC was performed using B16-F10 melanoma in mice (Adlego, Stockholm Sweden). 6 Study groups 1) Isotype IgG1 (MOPC21, BioXcell), 2) 7G12, 3) 7G12-DM1, 4) 7G12-DM1 & anti-PD1, 5) Isotype-DM1, 6) anti-PD1 (RPM1-14, BioXcell). Group 1 had 10 animals and the other 8 in each. 150 ug of antibody was administered i.v. for each of the 4 doses, twice weekly. Tumor size was measured by calipering during the study and at termination of the study tumors where excised and weighed.

(50) Results

(51) Tumor Growth of B16-F10 Melanoma, 7G12-Study

(52) At the end of the study, tumor sizes were statistically significantly smaller in weight compared to isotype control in the 7G12-DM1 group and even smaller when combined with PD1 inhibiting antibody, see FIG. 9 and Table 1.

(53) TABLE-US-00001 TABLE 1 Ttest vs. Ttest vs tumor (g) TGI isotype aPD1 avg stdev (T/C) p-value p-value Isotype 0.786 0.298 7G12 0.965 0.449 122% 0.34448 7G12-DM1 0.344 0.277 44% 0.00662 7G12-DM1 & aPD1 0.144 0.079 18% 0.00004 0.01813 Isotype-DM1 0.940 0.531 119% 0.46396 aPD1 0.346 0.184 44% 0.00420

Example 7—Activation of Antigen-Presenting Cells by Anti-CLPTM1 Antibodies Conjugated to DM1

(54) We have shown that dendritic cells have high CLPTM1 in the tumour microenvironment. Upon treatment with 7G12-DM1 in the B16 melanoma study in Example 6 we see an increase in PDL1 expression on plasmacytoid dendritic cells isolated from the tumour, indicating a higher level of immune activation (see FIG. 10). TILs were isolated and analysed as described in Example 5. Up-regulated PDL1 expression is an established marker for immune stimulation. This is one of the effects by which our ADC therapy is active.

Example 8-7G12-DM1 Affects Cytokine/Chemokines

(55) The 7G12-DM1 ADC positively affects chemokines capable of attracting immune cells in vivo. Sera were collected from the animals in the B16 melanoma study in Example 6 above and analysed by an Olink Proteomics prototype mouse panel (Uppsala Sweden). The ADC showed statistically significant increases in CCL2 (monocyte and dendritic cell attractant), CCL3 (macrophage and monocyte attractant), CCL5 (T-cell attractant), and CXCL9 (T-cell attractant) (see FIG. 11). PD1 inhibition alone did not affect these chemokines but did show an additive positive effect together with the 7G12-DM1 ADC. Also, a small tumour size at study termination correlated with high levels of these chemokines. Even within a treatment group (ADC and ADC&PD1 combination), CCL5 correlates with small tumour size illustrating an immune cell based anti-tumour response of variable degree between individual animals, FIG. 11. These chemokines may be used clinically to monitor response of therapy by measuring blood levels of these proteins.

Example 9—Tumour Growth of B16-F10 Melanoma Using a Fully Human Antibody 59D04 IgG2a

(56) ADC Preparation

(57) DM1-antibody conjugates were prepared as described in Example 3 but using a PEG4 linker in the SMCC in order to improve water solubility of the conjugate and scaled up to 40 mg of CLPTM1 antibody 59D04 IgG2a. A drug antibody ratio of 3.7 was achieved for the 59D04-DM1 conjugate.

(58) In Vivo Models

(59) A B16-F10 efficacy study was performed by Oncodesign (Montreal, Canada). 5 groups with 8 animals in each comprised: 1) Isotype IgG2a (C1.18.4 BioXcell). 2) 59D04-DM1, 3) 59D04-DM1&aPD1 (RPM1-14, BioXcell), 4) aPD1, and 5) 59D04. Six doses where given at 100 ug i.v. each twice weekly for three weeks.

(60) Antibody 59D04 is a fully human phage display derived antibody developed at Yumab (Germany) using a peptide target portion of CLPTM1 (LWRWQLYAAQSTKSPWNFLGDELYEQSDEEQDSVKVALLETNP)(SEQ ID NO: 61) and screening on a naive antibody library. In vivo data generated using 59D04 was using a scFv-mouse IgG2a Fc version produced and purified by Icosagen (Estonia) in CHO cells.

(61) Data in FIG. 12 shows a significant effect using the combination of 59D04-DM1 and anti-PD1 on tumour growth over isotype control. A summary of the data at day 20 of the study and p-value of a two-tailed T-test is shown in Table 2.

(62) TABLE-US-00002 TABLE 2 Ttest for tumor size difference at day 20 m{circumflex over ( )}3 m{circumflex over ( )}3 vs isotype vs PD1 avg stdev p-value p-value Isotype 941 208 59D04-DM1 840 374 0.6335 59D04-DM1 & aPD1 485 292 0.0439 0.1811 aPD1 883 482 0.8282 59D04 1167 935 0.6533

Example 10—Direct Activating Effect on Antigen Presenting Cells In Vitro by an Anti-CLPTM1 ADC

(63) Tubulin inhibitors can enhance the surface expression of T-cell co-stimulatory proteins CD80 and CD86. Both CD80 and CD86 are critical for cytotoxic T-cell activation. In vitro we derived M2 macrophages from a human donor using the protocol from Example 1 above. CLPTM1 expression was verified by flow cytometry using 59D04 antibody and shown in FIG. 13A. Isotype control staining (left peak) and staining with the 59D04 antibody (right peak) are shown.

(64) On these same M2 macrophages, a dilution series of either Isotype control, none, 59D04 mouse IgG2a or 59D04-DM1 was added and incubated overnight. CD80 and CD86 expression was analyzed by flow cytometry and found to be increased only with the DM1 conjugated antibody (FIGS. 13B and C). No enhancement was seen with an isotype control antibody or the non-conjugated 569D04 mAb. These data illustrate the applicability of our antibody drug conjugate comprising an anti-CLPTM1 antibody coupled to a tubulin inhibitor to stimulate antigen presenting cells in the tumor microenvironment and thereby enhance tumor cell killing by the immune system.

Example 11—Antibody Internalization into Cells for ADC

(65) Human CD14 cells were isolated according to standard protocols and cultured. A set of anti-CLPTM1 antibodies were coupled to phRodo (ThermoFisher). This dye fluoresces at low pH. Antibodies carrying phRodo that are internalized and transported to the lysosome compartments inside cells for protein degradation are thus transported to an environment of low pH making the dye brighter. The CLPTM1 antibody 59D04 as either mouse IgG1 or IgG2a chimera was conjugated and compared to an isotype control antibody and Santa Cruz G7. Flow cytometry data shown in FIG. 14 are from a 5 hour incubation. These antibodies capable of internalization and degradation by transport to endosomes/lysosomes are especially suited as antibody drug conjugates where the drug effect requires free drug to be released from the link to the antibody such as a covalent link between DM1 and the antibody.

Example 12—Anti-CLPTM1 Antibody Generation

(66) Mouse monoclonal antibody 7G12 was raised against a CLTPM1 synthetic peptide PWNFLGDELYEQSDE (SEQ ID NO: 40) using Rapid-Prime™ method by ImmunoPrecise (Victoria, Canada).

(67) Antibody 59D04 is a fully human phage display derived antibody developed at Yumab (Germany) using a peptide target portion of CLPTM1

(68) TABLE-US-00003 (SEQ ID NO: 61) (LWRWQLYAAQSTKSPWNFLGDELYEQSDEEQDSVKVALLETNP)
and screening on a naive antibody library.

(69) Variable heavy sequence of 59D04 is

(70) TABLE-US-00004 (SEQ ID NO: 59) EVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMHWVRQAPGKGLEWVAV ISYDGTNKYYADSVKGRFTIFRDNSKNTLYLQMNSLRAEDTAVYYCGSGS YWGQGTLVTVSS

(71) Variable light sequence of 59D04 is

(72) TABLE-US-00005 (SEQ ID NO: 60) QPVLTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMI YEVTNRPSGVSDRFSGSKSGNTASLTISGLQAEDEADYYCSSYKSSNTVV FGGGTKVTVL

(73) Variable heavy sequence of 7G12 (IgG1 isotype) is

(74) TABLE-US-00006 (SEQ ID NO: 62) QVQLQQSGTELVRPGTSVKVSCKASGYAFTNYLIEWVKQRPGQGLEWIGV INPGSGGTRYNEKFKGKATLTADKSSTTAHMQLSSLTSDDSAVYCARWGG NYSGYAMDYWGQGTSVTVSS

(75) Variable light sequence of 7G12 (Kappa isotype) is

(76) TABLE-US-00007 (SEQ ID NO: 63) QIVLTQSPVIMSASPGEKVTITCSASSSVSYMHWFQQKPGTSPKLWIYST SNLASGVPARFSGSGSGTSYSLTISRMEAEDAATYYCQQRSSYPPTFGAG TKL.

(77) The CDRs of the 7G12 antibody are shown in SEQ ID NOs: 66-71, and the CDRs for the 59D04 antibody are shown in SEQ ID NOs: 72-77.

(78) Consensus DNA sequences encoding the heavy and light chains for 7G12 are provided as SEQ ID NOs: 64 and 65, respectively.

Example 13—Antibody Affinity Determination

(79) Affinity of antibodies was determined by finding the half-maximal binding on the native target in a flow cytometry assay. Since the native target is found inside most cells and rarely on the cell surface, the assay was performed using plasma membrane-permeabilised cells, an intra-cellular staining protocol. Using the native target is more applicable when evaluating and comparing various antibodies as it is the true antigen to be bound in a therapeutic setting in vivo. 0-876 cells were permeabilised with BD-fix and permeabilisation buffer (554722 BD-Biosciences). Antibody at various concentrations was incubated with cells for 30 minutes at +4° C., then washed twice with wash buffer (BD-Biosciences 554723). Secondary antibodies a-mouse IgG-PE (Molecular Probes) was used for mouse monoclonal antibodies according to manufacturer's instructions and a-rabbit-IgG-PE (Molecular Probes) for rabbit monoclonal antibody (Abcam ERP8800). Three washes followed and fluorescence intensity in cells was quantified by FACS CantoII in PBS with cells in 2% FCS buffer.

(80) 7G12 had the greatest affinity showing more than having half maximal binding (Flow Signal) at 0.1 μg/mL suggesting a Kd (as defined by half maximal binding) below 1 nM. The assay was performed on O-876 cancer cell line. Binding data are shown in FIG. 15. Antibodies 7G12, 59D04 and the Abcam antibody showed more half maximal binding at 1 μg/mL.

(81) The Abcam antibody against CLPTM1 has reported affinity according to Biacore data supplied by the manufacturer of 0.0345 nM Kd. This was derived on a non-native recombinant portion of the antigen as test.

(82) Of these antibodies 59D04 showed internalisation (Example 11, FIG. 17). The Santa Cruz mAb was not internalised.

Example 14—4T1 Breast Cancer Model of Metastasis

(83) The syngeneic orthotopic breast cancer model 4T1 in mouse spontaneously metastasises to the lung. At the end of a study, the number of metastases can be counted. A study at Oncodesign (Montreal, Canada) was conducted using the same groups and treatments as for the B16 melanoma study in Example 9 using 59D04-DM1. Animals were treated with 100 ug i.v. of substances twice weekly for three weeks. At the end of the study, the groups had no statistically significant change in primary tumour size but the number of lung metastases was affected by both 59D04-mouse IgG2a-Fc and 59D04-DM1 compared to the isotype antibody control. Primary tumour sizes are plotted for each animal in FIG. 16A as well as the number of metastases counted in the lungs in FIG. 16B.

(84) Our cancer tissue microarray data has indicated that in general metastases have higher surface expression of CLPTM1 compared to primary tumours.

(85) Table 3 shows the significance of the treatment groups on lung metastases.

(86) TABLE-US-00008 TABLE 3 metastases p-value (2-sided Ttest) # of, average vs. isotype vs. aPD1 Isotype 55.5 59D04-DM1 17.6 0.000036 59D04-DM1 & aPD1 24.3 0.000188 0.078975 aPD1 41.6 0.077617 59D04 IgG2a 34.5 0.023511