[001] This application claims the benefit under 35 USC §119(e) from U.S. Provisional patent application serial number 62/416,968, filed November 3, 2016, which is incorporated herein by reference in its entirety.

Field

[002] This disclosure relates to methods and uses of a drug that blocks the

CD47/SIRPa interaction. More particularly, the disclosure relates to methods and uses that, in combination, are useful for improving cancer therapy.

Background

[003] Cancer cells are targeted for destruction by antibodies that bind to cancer cell antigens, and through recruitment and activation of macrophages by way of Fc receptor binding to the Fc portion of that antibody. Binding between CD47 on cancer cells and SIRPa on macrophages transmits a "don't eat me" signal that enables many tumour cells to escape destruction by macrophages. It has been shown that inhibition of the CD47/SIRPa interaction (CD47 blockade) will allow macrophages to "see" and destroy the target CD47+ cancer cell. The use of SIRPa to treat cancer by CD47 blockade is described in WO2010/130053.

[004] Trillium Therapeutics' WO2014/094122 describes a protein drug that inhibits the interaction between CD47 and SIRPa. This CD47 blockade drug is a form of human SIRPa that incorporates a particular region of its extracellular domain linked with a particularly useful form of an IgGl -based Fc region. In this form, the SIRPaFc drug shows dramatic effects on the viability of cancer cells that present with a CD47+ phenotype. The effect is seen particularly on acute myelogenous leukemia (AML) cells, and many other types of cancer. A soluble form of SIRP having significantly altered primary structure and potent CD47 binding affinity is described in WO2013/109752.

[005] Other CD47 blockade drugs have been described, and these include various

CD47 antibodies (see for instance Stanford's US8562997, and InhibRx' WO2014/123580), each comprising different antigen binding sites but having, in common, the ability to compete with endogenous SIRPa for binding to CD47, to interact with macrophages and, ultimately, to increase CD47+ disease cell depletion. These CD47 antibodies have activities in vivo that are quite different from those intrinsic to drugs that incorporate SIRPa structure. The latter, for instance, display negligible binding to red blood cells whereas the opposite property in CD47 antibodies, and in high affinity SIRPa variants, creates a need for strategies that accommodate a drug "sink" that follows administration.

[006] Still other agents are proposed for use in blocking the CD47/SIRPa axis.

These include CD47Fc proteins described in Viral Logic's WO2010/083253, and SIRPa antibodies as described in University Health Network's WO2013/056352, Eberhard's US 6913894, and elsewhere.

[007] The CD47 blockade approach in anti-cancer drug development shows great promise. There is a need to provide methods and means for improving the effect of these drugs, and in particular for improving the effect of the CD47 blockade drugs that incorporate CD47-binding forms of SIRPa.

Summary

[008] It is now shown that the anti-cancer effect of CD47 blockade therapy is improved when combined with an agent that inhibits histone deacetylase (HDAC) activity. More particularly, significant improvement in cancer cell vitality and/or depletion is seen when CD47+ cancer cells are treated with a CD47 blockade drug, such as a SIRPa-based drug or an anti-CD47 antibody, in combination with an HDAC inhibitor and particularly with romidepsin and related compounds. The two drugs cooperate in their effects on cancer cells, and cause the depletion of more cancer cells than can be accounted for by their individual effects. In related terms, the HDAC inhibitor is effective to enhance the anti-cancer activity of the CD47 blockade drug.

[009] In one aspect, there is provided a method for treating a subject presenting with CD47+ disease cells, comprising administering a combination comprising a CD47- binding form of SIRPa and an HDAC inhibitor, such as romidepsin.

[0010] In a related aspect, there is provided the use of a CD47 blockade drug, such as a SIRPa-based drug, in combination with an HDAC inhibitor for the treatment of a subject presenting with CD47+ disease cells such as cancer.

[0011] In another aspect there is provided a pharmaceutical combination comprising a CD47 blockade drug and HDAC inhibitor for use in the treatment of CD47+ disease cells.

[0012] There is also provided, in another aspect, a kit comprising a pharmaceutical combination comprising a CD47 blockade drug, such as a soluble SIRPa-based drug, and an HDAC inhibitor, together with instructions teaching their use in the treatment method herein described of CD47+ disease cells.

[0013] In a specific embodiment, the combination of the CD47 blockade drug and

HDAC inhibitor is for use in the treatment of cancer, including a blood cancer such as a myeloma, a lymphoma or a leukemia.

[0014] Other features and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples while indicating preferred embodiments of the disclosure are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description.

Brief Reference to the Drawings

[0015] Figure 1 shows results when the multiple myeloma cell lines MMls and

H929 are cultured in the presence of the HDAC inhibitor Romidepsin (at 1, 5 or 10 nM) for 48 hours. Cells are then washed; macrophages and SIRPaFc [TTI-621] (at 1, 5 or 100 nM) or Control Fc are added and the mixture is then subjected to the phagocytosis assay described below. As shown in Figure 1, culturing MMls (A) and H929 (B) in Romidepsin for 48 hours results increased SIRPaFc-mediated phagocytosis.

[0016] Figure 2 shows additional results when the multiple myeloma cell lines MMl.S and the cutaneous T-cell lymphoma line HH are cultured in the presence of the HDAC inhibitor Romidepsin (10 nM) for 48 hours. Cells are then washed; macrophages and 100 nM SIRPaFc (IgGl), SIRPaFc (IgG4), SIRPaFc (CV1) (IgG4 mut), CD47 monoclonal antibody (mAb) or Control Fc are added and the mixture is then subjected to the phagocytosis assay described herein. As shown in Figure 2, culturing MMl.S (A) and HH (B) in Romidepsin for 48 hours results in increased phagocytosis mediated by a variety of CD47 blockade drugs.

Detailed Description

[0017] The present disclosure provides methods, uses, combinations and kits useful for treating subjects that present with disease cells that have a CD47+ phenotype. In this method, subjects receive a combination of a CD47 blockade drug which preferably is a CD47-binding form of SIRPa, and a histone deacetylase (HDAC) inhibitor. The effect of this combination is superior to the effects of either agent alone. This is a statistically significant effect, or benefit, that results particularly when the CD47 blockade drug is a CD47-binding SIRPaFc-based agent. The effect is also seen when the CD47 blockade drug is a CD47-binding antibody. The effect is pronounced when the CD47+ disease cells are CD47+ cancer cells and tumours.

[0018] In one aspect, there is provided a method for treating a subject with CD47+ disease cells, comprising administering an effective amount of a drug combination comprising a CD47 blockade drug and a HDAC inhibitor.

[0019] In a related aspect, there is provided a use of a CD47 blockade drug in combination with a HDAC inhibitor for the treatment of a subject with CD47+ disease cells.

[0020] In another aspect, there is provided a combination comprising a CD47 blockade drug and HDAC inhibitor for use in the treatment of a CD47+ disease.

[0021] In a further aspect, there is provided a kit comprising a combination comprising a CD47 blockade drug and HDAC inhibitor together with instructions for the use in the treatment of CD47+ disease cells.

[0022] The term CD47+ disease cells means cells having the phenotype CD47+ and are associated with a disease. Cells that are CD47+ can be identified using the methods disclosed herein. In one embodiment, the CD47+ disease cells are cancer cells.

[0023] As used herein, a CD47 blockade drug is a drug or agent that interferes with and dampens or blocks signal transmission that results when CD47 interacts with macrophage-presented SIRPa. Also included are CD47-binding agents that block interaction between SIRPa and CD47. CD47-binding forms of human SIRPa are the preferred CD47 blockade drugs for use in the combination herein disclosed. These drugs are based on the CD47-binding extracellular region of human SIRPa. They comprise at least a part of the extracellular region sufficient to confer effective CD47 binding affinity and specificity. So-called "soluble" forms of SIRPa, lacking the membrane anchoring component, are useful and are described in the literature and include those referenced in Novartis' WO 2010/070047, and Stanford's WO2013/109752, and Trillium Therapeutics' WO2014/094122. The CD47 blockade drug can also be a bispecific Fc fusion protein that includes a CD47 binding site.

[0024] In a preferred embodiment, the soluble form of SIRPa is an Fc fusion.

More particularly, the CD47 blockade drug suitably comprises a CD47-binding part of the human SIRPa protein, in a form fused directly, or indirectly, with an antibody constant region, or Fc (fragment crystallisable) Unless otherwise stated, the term "human SIRPa" as used herein refers to a wild type, endogenous, mature form of human SIRPa. In humans, the SIRPa protein is found in two major forms. One form, the variant 1 or VI form, has the amino acid sequence set out as NCBI RefSeq NP_542970.1 (residues 27-504 constitute the mature form). Another form, the variant 2 or V2 form, differs by 13 amino acids and has the amino acid sequence set out in GenBank as CAA71403.1 (residues 30- 504 constitute the mature form). These two forms of SIRPa constitute about 80% of the forms of SIRPa present in humans, and both are embraced herein by the term "human SIRPa". Also embraced by the term "human SIRPa" are the minor forms thereof that are endogenous to humans and have the same property of triggering signal transduction through CD47 upon binding thereto. The present disclosure is directed most particularly to the drug combinations that include the human SIRP variant 2 form, or V2.

[0025] In the present drug combination, useful SIRPaFc fusion proteins comprise one, such as only one, of the three so-called immunoglobulin (Ig) domains that lie within the extracellular region of human SIRPa. More particularly, the present SIRPaFc proteins incorporate at least residues 32-137 of human SIRPa (a 106-mer), which constitute and define the IgV domain of the V2 form according to current nomenclature. This SIRPa sequence, shown below, is referenced herein as SEQ ID No. l .

EELQVIQPDKSVSVAAGESAILHCTVTSLIPVGPIQWFRGAGPARELIYNQKEGHFP RVTTVSESTKRENMDFSISISNITPADAGTYYCVKFRKGSPDTEFKSGA [SEQ ID No. l]

[0026] In a preferred embodiment, the SIRPaFc fusion proteins incorporate the

IgV domain as defined by SEQ ID No. l, and additional, flanking residues contiguous within the SIRPa sequence. This preferred form of the IgV domain, represented by residues 31-148 of the V2 form of human SIRPa, is a 118-mer having SEQ ID No. 2 shown below:

EEELQVIQPDKSVSVAAGESAILHCTVTSLIPVGPIQWFRGAGPARELIYNQKEGHF PRVTTV S ES TKRENMDF SIS ISNITP AD AGTYYC VKFRKGS PDTEFKS GAGTELS VR AKPS [SEQ ID No.2]

[0027] Desirable SIRPa fusion proteins incorporate an Fc region that preferably also has effector function. Fc refers to "fragment crystallisable" and represents the constant region of an antibody comprised principally of the heavy chain constant region and components within the hinge region. An Fc component "having effector function" is an Fc component having at least some natural or engineered function, such as at least some contribution to antibody-dependent cellular cytotoxicity or some ability to fix complement. Also, the Fc will at least bind to Fc receptors. These properties can be revealed using assays established for this purpose. Functional assays include the standard chromium release assay that detects target cell lysis. By this definition, an Fc region that is wild type IgGl or IgG4 has effector function, whereas the Fc region of a human IgG4 mutated to alter effector function, such as by incorporation of an alteration series that includes Pro233, Val234, Ala235 and deletion of Gly236 (EU), is considered not to have effector function. In a preferred embodiment, the Fc is based on human antibodies of the IgGl isotype. The Fc region of these antibodies will be readily identifiable to those skilled in the art. In embodiments, the Fc region includes the lower hinge-CH2-CH3 domains.

[0028] In a specific embodiment, the Fc region is based on the amino acid sequence of a human IgGl set out as P01857 in UniProtKB/Swiss-Prot, residues 104-330, and has the amino acid sequence shown below and referenced herein as SEQ ID No.3: DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL PAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGK* [SEQ ID No.3]

[0029] Thus, in embodiments, the Fc region has either a wild type or consensus sequence of an IgGl constant region. In alternative embodiments, the Fc region incorporated in the fusion protein is derived from any IgGl antibody having a typical effector-active constant region. The sequences of such Fc regions can correspond, for example, with the Fc regions of any of the following IgGl sequences (all referenced from GenBank), for example: BAG65283 (residues 242-473), , BAC04226.1 (residues 247- 478), BAC05014.1 (residues 240-471), CAC20454.1 (residues 99-320), BAC05016.1 (residues 238-469), BAC85350.1 (residues 243-474), BAC85529.1 (residues 244-475), and BAC85429.1 (residues (238-469).

[0030] In the alternative, the Fc region can be a wild type or consensus sequence of an IgG2 or IgG3 sequence, examples thereof being shown below:

a human IgG2, for example:

APPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHN AKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKG QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDISVEWESNGQPENNYKTTPP MLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

(SEQ ID No.4), as comprised in P01859 of the UniProtKB/Swiss-Prot database;

a human IgG3, for example:

APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFKWYVDGVEVH NAKTKPREEQYNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKTK GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPP MLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGK (SEQ ID No. 5), as comprised in P01860 of the UniProtKB/Swiss-Prot database.

[0031] In other embodiments, the Fc region has a sequence of a wild type human

IgG4 constant region. In alternative embodiments, the Fc region incorporated in the fusion protein is derived from any IgG4 antibody having a constant region with effector activity that is present but, naturally, is significantly less potent than the IgGl Fc region. The sequences of such Fc regions can correspond, for example, with the Fc regions of any of the following IgG4 sequences: P01861 (residues 99-327) from UniProtKB/Swiss-Prot and CAC20457.1 (residues 99-327) from GenBank.

[0032] In a specific embodiment, the Fc region is based on the amino acid sequence of a human IgG4 set out as P01861 in UniProtKB/Swiss-Prot, residues 99-327, and has the amino acid sequence shown below and referenced herein as SEQ ID No.6: ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQF NWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKG LPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPP VLD S DGS FFLYSRLTVDKS RWQEGNVF S C S VMHEALtTNHYTQ KSLSLSLGK [SEQ ID No.6]

[0033] In embodiments, the Fc region incorporates one or more alterations, usually not more than about 10, e.g., up to 5 such alterations, including amino acid substitutions that affect certain Fc properties. In one specific and preferred embodiment, the Fc region incorporates an alteration at position 228 (EU numbering), in which the serine at this position is substituted by a proline (S ²²⁸ P), thereby to stabilize the disulfide linkage within the Fc dimer. Other alterations within the Fc region can include substitutions that alter glycosylation, such as substitution of Asn ²⁹⁷ by glycine or alanine; half-life enhancing alterations such as T L, T S, and T F as taught in US62777375, and many others. Particularly useful are those alterations that enhance Fc properties while remaining silent with respect to conformation, e.g., retaining Fc receptor binding.

[0034] In a specific embodiment, and in the case where the Fc component is an

IgG4 Fc, the Fc incorporates at least the s ²²⁸ P mutation, and has the amino acid sequence set out below and referenced herein as SEQ ID No. 7:

ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQF NWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKG LPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPP VLD S DGS FFLYSRLTVDKS RWQEGNVF S C S VMHE ALUNHYTQ KSLSLSLGK [SEQ ID No.7]

[0035] The CD47 blockade drug used in the combination is thus preferably a SIRP fusion protein useful to inhibit the binding of human SIRPa with human CD47, thereby to inhibit or reduce transmission of the signal mediated via SIRPa-bound CD47, the fusion protein comprising a human SIRPa component and, fused therewith, an Fc component, wherein the SIRPa component comprises or consists of a single IgV domain of human SIRPa V2 and the Fc component is the constant region of a human IgG having effector function.

[0036] In one embodiment, the fusion protein comprises a SIRPa component consisting at least of residues 32-137 of the V2 form of wild type human SIRPa, i.e., SEQ ID No. l . In a preferred embodiment, the SIRPa component consists of residues 31-148 of the V2 form of human SIRPa, i.e., SEQ ID No. 2. In another embodiment, the Fc component is the Fc component of the human IgGl designated P01857, and in a specific embodiment has the amino acid sequence that incorporates the lower hinge-CH2-CH3 region thereof i.e., SEQ ID No.3.

[0037] In a preferred embodiment, therefore, the SIRPaFc fusion protein is provided and used in a secreted dimeric fusion form, wherein the fusion protein incorporates a SIRPa component having SEQ ID No.1 and preferably SEQ ID No, 2 and, fused therewith, an Fc region having effector function and having SEQ ID No.3. When the SIRPa component is SEQ ID No. 1, this fusion protein comprises SEQ ID No.8, shown below:

EEELQVIQPDKSVSVAAGESAILHCTVTSLIPVGPIQWFRGAGPARELIYNQKEGHFPRV TT VSESTKRENMDFSISISNITPADAGTYYCVKFRKGSPDTEFKSGAGTELSVRAKPSDKTH TC PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN AKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP Q VYTLPP SRDELTKNQ VSLTCL VKGFYP SDIAVEWE SNGQPENNYKTTPP VLD SDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK* [SEQ ID No.8]

[0038] When the SIRPa component is SEQ ID No. 2, this fusion protein comprises SEQ ID No. 9, shown below:

EEELQVIQPDKSVSVAAGESAILHCTVTSLIPVGPIQWFRGAGPARELrYNQKEGHF PRVTTVSESTKRENMDFSISISNITPADAGTYYCVKFRKGSPDTEFKSGAGTELSVR AKPSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPGK [SEQ ID No.9]

[0039] In alternative embodiments, the Fc component of the fusion protein is based on an IgG4, and preferably an IgG4 that incorporates the S ²²⁸ P mutation. In the case where the fusion protein incorporates the preferred SIRPa IgV domain of SEQ ID No.2, the resulting IgG4-based SIRPa-Fc protein has SEQ ID No. 10, shown below:

EEELQVIQPDKSVSVAAGESAILHCTVTSLIPVGPIQWFRGAGPARELrYNQKEGHF PRVTTVSESTKRENMDFSISISNITPADAGTYYCVKFRKGSPDTEFKSGAGTELSVR AKPSESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDP EVQFNWYVDGVEVFINAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKGLP S SIEKTIS KAKGQPREPQ VYTLPP S QEEMTKNQ VS LTCLVKGFYP S DI AVE WESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHN HYTQKSLSLSLGK [SEQ ID No.10]

[0040] In preferred embodiment, the fusion protein comprises, as the SIRPa IgV domain of the fusion protein, a sequence that has/comprises SEQ ID No.2. A preferred SIRPaFc is SEQ ID No.9. Another preferred SIRPaFc has/comprises SEQ ID No.10. The SIRPa sequence incorporated within the CD47 blockade drug can be varied, as described in the literature. That is, useful substitutions within SIRPa include one or more of the following: L ⁴ V/I, V ⁶ I/L, A ²¹ V, V ²⁷ I/L, ¹³¹ T/S/F, E ⁴⁷ V/L, K ⁵³ R, E ⁵⁴ Q, H ⁵⁶ P/R, S ⁶⁶ T/G, K ⁶⁸ R, V ⁹² I, F ⁹⁴ V/L, V ⁶³ I, and/or F ¹⁰³ V. In embodiments, these variants can incorporate a set of amino acid substitutions, such as V ⁶ I + V ²⁷ I + l ³¹ F + E47V + K ⁵³ R + E54Q + H ⁵⁶ P +S T + V ⁹² I .CD47-binding SIRPa variants of this type can be used either per se or as Fc fusions. [0041] In embodiments, the CD47 blockade drug is a variant of human SIRPa having higher binding affinity for human CD47 than wild type SIRPa. In a specific embodiment, the variant SIRPa has the sequence shown in SEQ ID No. 11 :

EEELQIIQPDKSVSVAAGESAILHCTITSLFPVGPIQWFRGAGPARVLIYNQRQGPF P RVTTVSETTKRENMDFSISISNITPADAGTYYCIKFRKGSPDTEFKSGAGTELSVRA KP [SEQ ID No. 11]

[0042] This SIRPa variant comprises the following amino acid substitutions relative to wild type SIRPa:

V ⁶ I + V ²⁷ I + l ³¹ F + E ⁴⁷ V + K ⁵³ R + E ⁵⁴ Q + H ⁵⁶ P +S ⁶⁶ T + V ⁹² I. In a specific embodiment, this variant SIRPa sequence can be fused with a mutated IgG4 Fc region including a Ser ²²⁸ Pro (EU) having virtually no effector function, to yield a CD47 blockade drug having the sequence shown in SEQ ID No. 12:

EEELQIIQPDKSVSVAAGESAILHCTITSLFPVGPIQWFRGAGPARVLIYNQRQGPFP RVTTVSETTKRENMDFSISISNITPADAGTYYCIKFRKGSPDTEFKSGAGTELSVRA KPSESKYGPPCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEV QFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNH YTQKSLSLSLGK* [SEQ ID No. 12]

[0043] Still other types of CD47 blockade drugs can be used in the present method and combination, instead of or in addition to the SIRPa-based drugs. These other drugs include particularly the CD47 antibodies, which bind to CD47 and antagonize the interaction with SIRPa. By blocking that interaction, and because of the Fc region of the antibody, the effect of the CD47 antibodies can be similar to the effect of the SIRPa-based Fc fusion drugs. Examples of CD47 antibodies are described in the literature such as Chugai's US2008/0107654; Stanford's WO2009/091601; InhibRx WO2013/119714, Celgene's WO2016/109415; and Janssen's WO2016/081423. Because these antibodies bind red blood cells, a dosing regimen that takes this into account has been developed and is described in WO2014/149477. The properties of a useful antibody include simply the ability to bind to CD47 in a way that ultimately inhibits signaling by SIRPa, i.e., as an antagonist.

[0044] In one embodiment, the CD47 blockade drug is an anti-CD47 antibody that is a chimeric, humanized, human or otherwise recombinant, monoclonal or polyclonal antibody based on the sequence of antibody B6H12 known from the literature and including the sequences:

[0045] Amino acid sequence of B6H12 heavy chain variable region

EVQLVESGGDLVKPGGSLKLSCAASGFTFSGYGMSWVRQTPDKRLEWVATITSGGTYTYY PDSVKGRFTISRDNAKNTLYLQIDSLKSEDTAIYFCARSLAGNAMDYWGQGTSVTVSS

[SEQ ID No.13]

[0046] Amino acid sequence of B6H12 light chain variable region

DIVMTQSPA LSV PGDRVSLSCRASQ ISDYLHWYQQKSHESPRLLIKFASQS ISGI PS RFSGSGSGSDF LS INSVEPEDVGVYYCQNGHGFPR FGGGTKLEIK [SEQ ID No.14]

[0047] A full sequence for this antibody and the CDR sequences therein, are available from Figure 1 in US21030142786, the entire contents of which are incorporated herein by reference.

[0048] Other CD47 blockade drugs include CD47Fc proteins, as taught by Viral

Logic in WO2010/083253 and by Stanford in US8377448), as well as SIRPa antibodies, as described in UHN's WO2013/056352, Stanford's WO2016/022971, Eberhard's US 6913894, and elsewhere. These particular CD47 blockade drugs are not ideal, because they bind to the SIRPa which is presented by most cells in the body. CD47 on the other hand is presented only on macrophages and on a limited variety of other cell types, thus limiting the effects of CD47 binding agents to those cells and tissues.

[0049] In a SIRPaFc fusion protein, the SIRPa component and the Fc component are fused, either directly or indirectly, to provide a single chain polypeptide that is ultimately produced as a dimer in which the single chain polypeptides are coupled through intrachain disulfide bonds formed within the Fc region. The nature of the fusing region is not critical. The fusion may be direct between the two components, with the SIRP component constituting the N-terminal end of the fusion and the Fc component constituting the C-terminal end. Alternatively, the fusion may be indirect, through a linker comprised of one or more amino acids, desirably genetically encoded amino acids, such as two, three, four, five, six, seven, eight, nine or ten amino acids, or any number of amino acids between 5 and 100 amino acids, such as between 5 and 50, 5 and 30 or 5 and 20 amino acids. A linker may comprise a peptide that is encoded by DNA constituting a restriction site, such as a BamHI, Clal, EcoRI, Hindlll, PstI, Sail and Xhol site and the like. [0050] The linker amino acids typically and desirably have some flexibility to allow the Fc and the SIRP components to adopt their active conformations. Residues that allow for such flexibility typically are Gly, Asn and Ser, so that virtually any combination of these residues (and particularly Gly and Ser) within a linker is likely to provide the desired linking effect. In one example, such a linker is based on the so-called G ₄ S sequence (Gly-Gly-Gly-Gly-Ser) (SEQ ID No. 15) which may repeat as (G ₄ S) _n where n is 1, 2, 3 or more, or is based on (Gly)n, (Ser)n, (Ser-Gly)n or (Gly-Ser)n and the like. In another embodiment, the linker is GTELSVRAKPS (SEQ ID No.16). This sequence constitutes SIRPa sequence that C-terminally flanks the IgV domain (it being understood that this flanking sequence could be considered either a linker or a different form of the IgV domain when coupled with the IgV minimal sequence described above). It is necessary only that the fusing region or linker permits the components to adopt their active conformations, and this can be achieved by any form of linker useful in the art.

[0051] As noted, the CD47 blockade drug such as a SIRPaFc fusion is useful to inhibit interaction between SIRPa and CD47, thereby to block signalling across this axis. Stimulation of SIRPa on macrophages by CD47 is known to inhibit macrophage-mediated phagocytosis by deactivating myosin-II and the contractile cytoskeletal activity involved in pulling a target into a macrophage. Activation of this cascade is therefore important for the survival of CD47+ disease cells, and blocking this pathway enables macrophages to eradicate or at least reduce the CD47+ disease cell population.

[0052] The term "CD47+" is used with reference to the phenotype of cells targeted for binding by the present CD47 blockade drugs. Cells that are CD47+ can be identified by flow cytometry using CD47 antibody as the affinity ligand. CD47 antibodies that are labeled appropriately are available commercially for this use (for example, the antibody product of clone B6H12 is available from BD Biosciences). The cells examined for CD47 phenotype can include standard tumour biopsy samples including particularly blood samples taken from the subject suspected of harbouring endogenous CD47+ cancer cells. CD47 disease cells of particular interest as targets for therapy with the present drug combination are those that "over-express" CD47. These CD47+ cells typically are disease cells, and present CD47 at a density on their surface that exceeds the normal CD47 density for a cell of a given type. CD47 overexpression will vary across different cell types, but is meant herein to refer to any CD47 level that is determined, for instance by flow cytometry or by immunostaining or by gene expression analysis or the like, to be greater than the level measurable on a counterpart cell having a CD47 phenotype that is normal for that cell type.

[0053] The present drug combination comprises both a CD47 blockade drug that preferably comprises a soluble form of a SIRPa-based drug, as just described, and an inhibitor of histone deacetylase.

[0054] Histone deacetylases (HDACs) are a group of enzymes that facilitates the removal of acetyl groups (deacetylation) from lysine residues on histone and non-histone proteins. HDACs regulate chromatin structure and transcription by erasing acetyl groups on histones. Deacetylation of non-histone proteins regulates a range of cellular processes. Human have 18 known HDACs divided in to 4 classes (Class I, II, III and IV).

[0055] HDAC inhibitors interfere with the deacetylation process, by inhibiting the enzyme activity of HDACs. These have been described as "epigenetic modifiers" because of their regulation of post-translational modifications. The anti-tumor mechanisms of HDACi are not fully understood, but have been shown to affect a wide range of cellular processes, for example leading to the induction of cell death, cell cycle arrest, and differentiation and inhibition of invasion and migration. HDAC inhibitors are divided into at least four structural classes: hydroxamates, cyclic peptides (depsipeptides), aliphatic acids and benzamides. The selectivity of the compounds varies. Pan-HDACi (nonselective), class-selective and isotype selective inhibitors have been described, resulting in different, often non-redundant anti-tumor effects and target specificity.

[0056] In embodiments, the HDAC inhibitor used in the drug combination is a depsipeptide. These are non-ribosomal peptides cyclized via an ester bond. They can contain non-protein amino acids. They include the cyclotetrapeptides that include apicidin and romidepsin, as well as Spiruchostatin A, etamycin, papuamide, neamphamide A, callipeltin A and mirabamides A-D. In particular embodiments, the HDAC inhibitor used in the pharmaceutical combination is a depsipeptide that inhibits the enzymatic activity of HDACs belonging to Class I, such as HDACI, HDAC2, HDAC3 and HDAC 8.. These

[0057] In a preferred embodiment, the HDAC inhibitor in the present combination is romidepsin, a depsipeptide that is a potent inhibitor of Class I HDACs, having the structure shown below:

Other HDAC inhibitors include compounds such as those listed below

[0059] In a specific embodiment of the present method, the CD47 blockade drug is used in combination with romidepsin. As noted, romidepsin is marketed under the trademark Istodax® and is provided as a lyophilized powder for intravenous injection. It is supplied as a kit which includes a sterile, lyophilized powder in a single-use vial containing 10 mg of romidepsin and 20 mg of carrier, povidone. Each kit also provides one sterile vial containing 2 mL of diluent (80% propylene glycol, and 20% dehydrated alcohol), for reconstitution in 0.9% saline. Established dosing is 14mg/m2 with intravenous administrations on days 1, 8 and 15 of a 28 day cycle. It can be used in this same manner for purposes of the present disclosure, although cooperation with the CD47 blockade drug should permit the use of a reduced romidepsin dose or dosing frequency. It is used particularly for the treatment of blood cancers such as forms of T cell lymphoma including peripheral and cutaneous, and can be used for this purpose when combined with CD47 blockade drug.

[0060] Each drug included in the combination can be formulated separately for use in combination. The drugs are said to be used "in combination" when, in a recipient of both drugs, the effect of one drug enhances or at least influences the effect of the other drug.

[0061] The two drugs in the combination cooperate to provide an effect on target

CD47+ cells that is greater than the effect of either drug alone. This benefit manifests as a statistically significant improvement in a given parameter of target cell fitness or vitality. For instance, a benefit in CD47+ cancer cells, when exposed to a combination of CD47 blockade drug and HDAC inhibitor, could be a statistically significant decrease in the number of living cancer cells (hence a depletion), relative to non-treatment, or a decrease in the number or size of cancer cells or tumours, or an improvement in the endogenous location or distribution of any particular tumour type. In embodiments, the improvement resulting from treatment with the drug combination can manifest as an effect that is at least additive and desirably synergistic, relative to results obtained when only a single agent or an agent combination is used.

[0062] In use, each drug in the combination can be formulated as it would be for monotherapy, in terms of dosage size and form and regimen. In this regard, the improvement resulting from their combined use may permit the use of somewhat reduced dosage sizes or frequencies, as would be revealed in an appropriately controlled clinical trial.

[0063] The mechanism by which an HDAC inhibitor contributes to the activity of a CD47 blockade drug, in the present combination, is not known. The HDAC inhibitors likely have a direct activity on some tumour cells, and preliminary data suggest that treatment of tumor cells with these inhibitors results in upregulation of pro-phagocytic ("eat-me") signals on the surface of some tumor cells. [0064] In this approach, each drug is provided in a dosage form comprising a pharmaceutically acceptable carrier, and in a therapeutically effective amount. As used herein, "pharmaceutically acceptable carrier" means any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible and useful in the art of protein/antibody formulation. Examples of pharmaceutically acceptable carriers include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like, as well as combinations thereof. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Pharmaceutically acceptable carriers may further comprise minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the pharmacological agent. Each of the CD47 blockade drug, e.g., SIRPaFc fusion protein, and the HDAC inhibitor is formulated using practises standard in the art of therapeutics formulation. Solutions that are suitable for intravenous administration, such as by injection or infusion, are particularly useful. The HDAC inhibitor will of course be formulated as permitted by the regulatory agencies when it is a drug approved for use in humans.

[0065] Sterile solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients noted above, as required, followed by sterilization microfiltration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation are vacuum drying and freeze-drying (lyophilization) that yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0066] As used herein, "effective amount" refers to an amount effective, at dosages and for a particular period of time necessary, to achieve the desired therapeutic result. A therapeutically effective amount of each drug in the combination may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the drug to elicit a desired response in the recipient. A therapeutically effective amount is also one in which any toxic or detrimental effects of the pharmacological agent are outweighed by the therapeutically beneficial effects. The HDAC inhibitor will of course be formulated in amounts that are suitable for patient dosing, as permitted by the regulatory agencies that have approved its use in humans. In use, each drug in the combination thus is formulated as it would be for monotherapy, in terms of dosage size and form and regimen. In this regard, the cooperation/benefit resulting from their combined use may permit the use of somewhat reduced dosage sizes or frequencies, as would be revealed in an appropriately controlled clinical trial.

[0067] The SIRPaFc fusion protein can be administered to the subject through any of the routes established for protein delivery, in particular intravenous, intradermal, intratumoural and subcutaneous injection or infusion, or by oral or nasal administration.

[0068] The drugs in the present combination can be administered sequentially or, essentially at the same time. In embodiments, the HDAC inhibitor is given before administration of the CD47 blockade drug, e.g., SIRPaFc. In the alternative, the HDAC inhibitor can be given after or during administration of the CD47 blockade drug, e.g., SIRPaFc. Thus, in embodiments, the subject undergoing therapy is a subject already treated with one of the combination drugs, such as the HDAC inhibitor, that is then treated with the other of the combination drugs, such as the CD47 blockade drug.

[0069] Dosing regimens are adjusted to provide the optimum desired response

(e.g., a therapeutic response). For example, a single bolus of each drug may be administered, or several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the therapeutic situation. It is especially advantageous to formulate parenteral compositions in unit dosage form for ease of administration and uniformity of dosage. "Unit dosage form" as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.

[0070] The drugs can be formulated in combination, so that the combination can be introduced to the recipient in one administration, e.g., one injection or one infusion bag. Alternatively, the drugs can be combined as separate units that are provided together in a single package, and with instructions for the use thereof according to the present method. In another embodiment, an article of manufacture containing the SIRPaFc drug and HDAC inhibitor combination in an amount useful for the treatment of the disorders described herein is provided. The article of manufacture comprises one or both drugs of the present antibody drug combination, as well as a container and a label. Suitable containers include, for example, bottles, vials, syringes, and test tubes. The containers may be formed from a variety of materials such as glass or plastic. The container holds a composition 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 vial having a stopper pierceable by a hypodermic injection needle). The label on or associated with the container indicates that the composition is used in combination with another CD47 blockade drug in accordance with the present disclosure, thereby to elicit a synergistic effect on the CD47+ disease cells. The article of manufacture may further comprise a second container comprising a pharmaceutically-acceptable buffer, such as phosphate- buffered saline, Ringer's solution and dextrose solution. It may further include other matters desirable from a commercial and use standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.

[0071] For administration the dose for the CD47 blockade drug will be within the range from about 0.0001 to 100 mg/kg, and more usually 0.01 to 5 mg/kg, of the host body weight. For example SIRPaFc dosages can be 0.3 mg/kg body weight, 1 mg/kg body weight, 3 mg/kg body weight, 5 mg/kg body weight or 10 mg/kg body weight or within the range of 0.1 -100 mg/kg. When the CD47 blockade drug is a SIRPaFc fusion protein of SEQ ID No.9, the dose can be about l-5mg per injection, such as intratumoural injection.

[0072] The SIRPaFc protein displays negligible binding to red blood cells. There is accordingly no need to account for an RBC "sink" when dosing with the drug combination. Relative to other CD47 blockade drugs that are bound by RBCs, it is estimated that the present SIRPaFc fusion can be effective at doses that are less than half the doses required for drugs that become RBC-bound, such as CD47 antibodies. Moreover, the SIRPa-Fc fusion protein is a dedicated antagonist of the SIRPa-mediated signal, as it displays negligible CD47 agonism when binding thereto. There is accordingly no need, when establishing medically useful unit dosing regimens, to account for any stimulation induced by the drug.

[0073] The drug combination is useful to treat a variety of CD47+ disease cells. These include particularly CD47+ cancer cells, including liquid and solid tumours. Solid tumours can be treated with the present drug combination, to reduce the size, number, distribution or growth rate thereof and to control growth of cancer stem cells. Such solid tumours include CD47+ tumours in bladder, brain, breast, lung, colon, ovary, prostate, liver and other tissues as well. In one embodiment, the drug combination can used to inhibit the growth or proliferation of hematological cancers. As used herein, "hematological cancer" refers to a cancer of the blood, and includes leukemia, lymphoma and myeloma among others. "Leukemia" refers to a cancer of the blood, in which too many white blood cells that are ineffective in fighting infection are made, thus crowding out the other parts that make up the blood, such as platelets and red blood cells. It is understood that cases of leukemia are classified as acute or chronic. Certain forms of leukemia may be, by way of example, acute lymphocytic leukemia (ALL); acute myeloid leukemia (AML); chronic lymphocytic leukemia (CLL); chronic myelogenous leukemia (CML); myeloproliferative disorder/neoplasm (MPDS); and myelodysplasia syndrome. "Lymphoma" may refer to a Hodgkin's lymphoma, both indolent and aggressive non- Hodgkin's lymphoma, cutaneous T cell lymphoma (CTCL), Burkitt's lymphoma, Mantle cell lymphoma (MCL) and follicular lymphoma (small cell and large cell), among others. Myelomas include multiple myeloma (MM), giant cell myeloma, heavy-chain myeloma, and light chain myeloma and Bence-Jones myeloma.

[0074] In some embodiments, the hematological cancer treated with the drug combination is a CD47+ leukemia, preferably selected from acute lymphocytic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, and myelodysplastic syndrome, preferably, human acute myeloid leukemia.

[0075] In other embodiments, the hematological cancer treated with the drug combination is a CD47+ lymphoma or myeloma selected from Hodgkin's lymphoma, both indolent and aggressive non-Hodgkin's lymphoma, diffuse large cell lymphoma (DLBCL), mantle cell lymphoma, T cell lymphoma including mycosis fungoides, Sezary's syndrome, Burkitt's lymphoma, follicular lymphoma (small cell and large cell), multiple myeloma (MM), giant cell myeloma, heavy-chain myeloma, and light chain or Bence- Jones myeloma as well as leimyosarcoma.

[0076] In a specific embodiment, the cancer treated with the present combination is multiple myeloma. In another specific embodiment, the targeted cancer is mantle cell lymphoma. In a further embodiment, the cancer treated with the present combination is relapsed or refractory Hodgkin's lymphoma. In another specific embodiment, the CD47 blockade drug is SIRPaFc. In a further specific embodiment the HDAC inhibitor is romidepsin. [0077] In still other embodiments, romidepsin is used in combination with

SIRPaFc, such as SEQ ID No.9 or SEQ ID No.10, such as for the treatment of cutaneous T cell lymphoma or multiple myeloma. In another embodiment, the combination is used to treat a T cell lymphoma such as mycosis fungoides or Sezary's syndrome.

[0078] Thus, in specific embodiments, there is provided the use of a CD47 blockade drug in combination with an HDAC inhibitor for the treatment of a particular CD47+ cancer, wherein:

i) the CD47 blockade drug is SIRPaFc of SEQ ID No.9 and the HDAC inhibitor is romidepsin, such as for the treatment of a cancer that is cutaneous T cell lymphoma or multiple myeloma or relapsed or refractory Hodgkin's lymphoma.;

ii) the CD47 blockade drug is SIRPaFc of SEQ ID No.10 and the HDAC inhibitor is romidepsin, such as for the treatment of a cancer that is cutaneous T cell lymphoma or multiple myeloma or relapsed or refractory Hodgkin's lymphoma.;

iii) the CD47 blockade drug is anti-CD47 antibody and the HDAC inhibitor is romidepsin, such as for the treatment of a cancer that is cutaneous T cell lymphoma or multiple myeloma.

[0079] It will be appreciated that other CD47 blockade drugs can be used in combination with HDAC inhibitors. Desirable combinations will show a statistically significant improvement in cancer cell response. This can be demonstrated as a statistically significant improvement in HDAC inhibitor activity caused by combination with a CD47 blockade drug, or vice versa, where statistical significance is shown as noted in the examples that follow and desirably, provides a p value >0.05 and more desirably >0.01 such as >0.001.

[0080] The combination therapy, comprising CD47 blockade and HDAC inhibition can also be exploited together with any other agent or modality useful in the treatment of the targeted indication, such as surgery as in adjuvant therapy, or with additional chemotherapy as in neoadjuvant therapy.

[0081] The following non-limiting examples are illustrative of the present disclosure.

Examples

[0082] Heparinized whole blood was obtained from normal healthy human donors

(Biological Specialty Corporation) and informed consent was obtained from all donors. Peripheral blood mononuclear cells (PBMCs) were isolated over Ficoll-Paque Plus density gradient (GE Healthcare) and CD 14+ monocytes were isolated from PBMCs by positive selection using CD14 antibody-coated MicroBead separation (Miltenyi Biotec). Monocytes were differentiated into macrophages by culturing for seven days in X-Vivo-15 media (Lonza) supplemented with M-CSF (PeproTech). 24 hours prior to the phagocytosis assay, macrophages were primed with IFN-γ (PeproTech). 48 hours prior to the phagocytosis assay, Romidepsin (1, 5 or 10 nM) was added to tumor cells. On the day of the phagocytosis assay, macrophages were co-cultured with a violet proliferation dye 450 (VPD450)-labeled human multiple myeloma cell lines (MMls or H929) in the presence of 1, 5 or 100 nM human SIRPaFc (V region of human SIRPa variant 2 fused with IgGl Fc), 100 nM control Fc [human IgGl Fc region (hinge-CH2-CH3)] for two hours. Phagocytosis was assessed as % VPD450+ cells of live, single CD14+CDl lb+ macrophages by flow cytometry. Results shown in Figures 1 and 2 are representative of two independent experiments.

[0083] Results are shown in Figure 1 and are duplicates from a single experiment. Results shown in Figure 2 reveal the effects of a wider array of CD47 blockade drugs used in combination with romidepsin. For Figure 2, methods were the following:

[0084] Macrophages were prepared from human peripheral blood mononuclear cells (PBMCs) obtained from healthy donors (BioreclamationlVT); informed consent was obtained from all donors. CD14 ⁺ monocytes were isolated by positive selection using the EasySep® human monocyte isolation kit (Stemcell Technologies). Monocytes were differentiated into macrophages by culturing the cells in X-VIVO 15 media (Lonza) supplemented with human m-CSF (PeproTech) for 10 days. Macrophages were primed with human IFNy (PeproTech) one day prior to the phagocytosis assay. 48 hours prior to the phagocytosis assay, lOnM Romidepsin was added to tumor cells. On the day of the phagocytosis assay, macrophages were co-cultured with a violet proliferation dye 450 (VPD450)-labeled human cell lines (MM1.S or HH) in the presence 100 nM human SIRPaFc (IgGl, SEQ ID No.9), SIRPaFc (IgG4,SEQ ID No.10), SIRPaFc (CVl,(IgG4 mut, SEQ ID No. 12), CD47 monoclonal antibody B6H12 (mAb, SEQ ID Nos. 13 and 14) or Control Fc for two hours. Phagocytosis was assessed as % VPD450+ cells of live, single CD14+CDl lb+ macrophages by flow cytometry. Statistical significance was calculated by unpaired t-test using GraphPad Prism software comparing untreated vs romidepsin treated cells. [0085] While the present disclosure has been described with reference to what are presently considered to be the preferred examples, it is to be understood that the disclosure is not limited to the disclosed examples. To the contrary, the disclosure is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims.

[0086] All publications, patents and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety.