Patent Application
20220323600
The present application is filed with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled “2020-04-09_01131-0026-00PCT_ST25.txt” created on Apr. 9, 2020, which is 298,657 bytes in size. The information in the electronic format of the sequence listing is incorporated herein by reference in its entirety.
This application relates to targeted T-cell engaging agents (TEACs) and antibody tumor-targeting assembly complexes (ATTACs) for treating cancer.
The TEAC or ATTAC described herein may have, for example, longer half-life or comprise multiple components in a single agent.
Cancer creates significant loss of life, suffering, and economic impact. Immunotherapeutic strategies for targeting cancer have been an active area of translational clinical research.
A variety of other approaches have been explored for immunotherapy, but many of these prior approaches lack sufficient specificity to particular cancer cells. For example, demibodies have been designed each having an scFv portion binding to different antigens on a target cell, an Fc domain allowing pairing to a complementary demibody, and a binding partner capable of forming an association to another binding partner on a complementary demibody. WO 2007/062466. These demibodies, however, are not necessarily specific to cancer cells and could bind and have activity on other cells expressing the same antigens. See also WO 2013/104804, which provides a first polypeptide with a targeting moiety binding to a first antigen and a first fragment of a functional domain, along with a second polypeptide with a targeting moiety binding to a second antigen and a second fragment of a functional domain that is complementary to the first fragment of the functional domain. Likewise, this approach is not necessarily specific to cancer cells and could bind and have activity on other cells expressing the same antigens.
Bispecific T-cell Engaging Antibodies (BiTEs) have been proposed by others; however, these constructs are often not sufficiently specific to the tumor environment. Further, current bi-specific antibodies activate T cells via CD3. Although not widely discussed, these agents are incredibly potent and are given at extremely low doses compared with whole antibody therapies. This will be partly due to the fact that these reagents can theoretically activate every T cell by binding to CD3. When someone has a viral infection, around 1-10% of their T cells are activated and they feel lethargic and ill because of the immune response. When more T cells are activated, this can lead to larger problems including cytokine release syndrome (CRS) and death in rare cases. CRS can be triggered by release of cytokines from cells targeted by biologics, as well as by cytokine release from recruited immune effector cells. Therefore, there is a need to limit the total number of T cells that are activated using these systems.
This application describes a TEAC or ATTAC comprising a half-life extending moiety. Agents or components with a half-life extending moiety may simplify dosing regimens and allow lower doses of agents to be administered to achieve the same efficacy as agents without a half-life extending moiety, or may reduce dosing frequency required to achieve efficacy. Further, a single-agent TEAC or ATTAC may facilitate methods of making these agents or components and simplify administration to one active agent.
This disclosure describes TEACs and ATTACs that comprise half-life extension moieties. These TEACs and ATTACs may be two-component kits or compositions or a single component of a kit or composition.
This application also describes single-agent TEACs and ATTACs.
This disclosure describes an agent for treating cancer in a patient comprising a first component comprising a targeted T-cell engaging agent comprising: a first targeting moiety that binds a tumor antigen expressed by the cancer; a first T-cell engaging domain capable of T-cell engaging activity when binding a second T-cell engaging domain, wherein the second T-cell engaging domain is not part of the first component, and wherein the first T-cell engaging domain comprises either a VH domain or VL domain;
a first inert binding partner for the first T-cell engaging domain binding to the first T-cell engaging domain such that the first T-cell engaging domain does not bind to the second T-cell engaging domain unless the inert binding partner is removed, wherein if the first T-cell engaging domain comprises a VH domain, the inert binding partner comprises a VL domain and if the first T-cell engaging domain comprises a VL domain, the inert binding partner comprises a VH domain;
a first half-life extending moiety, wherein the first half-life extending moiety is attached (directly or indirectly) to the first inert binding partner; and a protease cleavage site separating the first T-cell engaging domain and the first inert binding partner, wherein the protease cleavage site is capable of releasing the inert binding partner and half-life extending moiety from the T-cell engaging domain in the presence of a protease (1) expressed by the cancer or in the cancer microenvironment or (2) colocalized to the cancer by a targeting moiety that binds a tumor antigen expressed by the cancer and that is the same or different from the targeting moiety in the agent, and
a second component comprising a targeted T-cell engaging agent comprising:
a second targeting moiety that binds a tumor antigen expressed by the cancer;
a second T-cell engaging domain capable of T-cell binding activity when binding a first T-cell engaging domain, wherein the first T-cell engaging domain is not part of the second component, and wherein the second T-cell engaging domain comprises either a VH domain or VL domain;
a second inert binding partner for the second T-cell engaging domain binding to the second T-cell engaging domain such that the second T-cell engaging domain does not bind to the first T-cell engaging domain unless the inert binding partner is removed, wherein if the second T-cell engaging domain comprises a VH domain, the inert binding partner comprises a VL domain and if the second T-cell engaging domain comprises a VL domain, the inert binding partner comprises a VH domain; and
a second half-life extending moiety, wherein the second half-life extending moiety is attached (directly or indirectly) to the second inert binding partner;
a protease cleavage site separating the second T-cell engaging domain and the second inert binding partner, wherein the protease cleavage site is capable of releasing the inert binding partner and half-life extending moiety from the T-cell engaging domain in the presence of a protease (1) expressed by the cancer or in the cancer microenvironment or (2) colocalized to the cancer by a targeting moiety that binds a tumor antigen expressed by the cancer and that is the same or different from the targeting moiety in the agent,
wherein the first and second T-cell engaging domains are capable of binding a T cell when neither is bound to an inert binding partner, and further wherein if the first T-cell engaging domain comprises a VH domain, the second T-cell engaging domain comprises a VL domain and if the first T-cell engaging domain comprises a VL domain, the second T-cell engaging domain comprises a VH domain.
This disclosure also describes an agent for treating cancer in a patient comprising:
a first component comprising a targeted immune cell engaging agent comprising:
a targeting moiety capable of targeting the cancer;
a first immune cell engaging domain capable of immune engaging activity when binding a second immune cell engaging domain, wherein the second immune cell engaging domain is not part of the first component, optionally wherein the first immune cell engaging domain is a T-cell engaging domain;
a first inert binding partner for the first immune cell engaging domain binding to the first immune cell engaging domain such that the first immune cell engaging domain does not bind to the second immune cell engaging domain unless the inert binding partner is removed, wherein if the first immune cell engaging domain comprises a VH domain, the inert binding partner comprises a VL domain and if the first immune cell engaging domain comprises a VL domain, the inert binding partner comprises a VH domain;
a first half-life extending moiety, wherein the first half-life extending moiety is attached (directly or indirectly) to the first inert binding partner; and
a protease cleavage site separating the first immune cell engaging domain and the first inert binding partner, wherein the protease cleavage site is capable of releasing the inert binding partner and half-life extending moiety from the immune cell engaging domain in the presence of a protease (1) expressed by the cancer or in the cancer microenvironment or (2) colocalized to the cancer by a targeting moiety that binds a tumor antigen expressed by the cancer and that is the same or different from the targeting moiety in the agent, and
a second component comprising a selective immune cell engaging agent comprising:
an immune cell selection moiety capable of selectively targeting an immune cell;
a second immune cell engaging domain capable of immune cell engaging activity when binding the first immune cell engaging domain, wherein the first and second immune cell engaging domains are capable of binding when neither is bound to an inert binding partner, optionally wherein the second immune cell engaging domain is a immune cell engaging domain;
a second inert binding partner for the second immune cell engaging domain binding to the second immune cell engaging domain such that the second immune cell engaging domain does not bind to the first immune cell engaging domain unless the inert binding partner is removed, wherein if the second immune cell engaging domain comprises a VH domain, the inert binding partner comprises a VL domain and if the second immune cell engaging domain comprises a VL domain, the inert binding partner comprises a VH domain; and
a second half-life extending moiety, wherein the second half-life extending moiety is attached (directly or indirectly) to the second inert binding partner;
a protease cleavage site separating the second immune cell engaging domain and the second inert binding partner, wherein the protease cleavage site is capable of releasing the inert binding partner and half-life extending moiety from the immune cell engaging domain in the presence of a protease (1) expressed by the cancer or in the cancer microenvironment or (2) colocalized to the cancer by a targeting moiety that binds a tumor antigen expressed by the cancer and that is the same or different from the targeting moiety in the agent,
wherein the first and second immune cell engaging domains are capable of binding an immune cell when neither is bound to an inert binding partner, and further wherein if the first immune cell engaging domain comprises a VH domain, the second immune cell engaging domain comprises a VL domain and if the first immune cell engaging domain comprises a VL domain, the second immune cell engaging domain comprises a VH domain.
This disclosure also describes a component for use in a kit or composition for treating cancer in a patient comprising a first targeted immune cell engaging agent comprising:
a targeting moiety that binds a tumor antigen expressed by the cancer;
an immune cell engaging domain capable of immune cell binding activity when binding another immune cell engaging domain, wherein the other immune cell engaging domain is not part of the first component, and wherein the immune cell engaging domain comprises either a VH domain or VL domain, optionally wherein the immune cell engaging domain is a T-cell engaging domain;
an inert binding partner for the immune cell engaging domain binding to the immune cell engaging domain such that the immune cell engaging domain does not bind to the other immune cell engaging domain unless the inert binding partner is removed, wherein if the immune cell engaging domain comprises a VH domain, the inert binding partner comprises a VL domain, and if the immune cell engaging domain comprises a VL domain, the inert binding partner comprises a VH domain;
a half-life extending moiety, wherein the half-life extending moiety is attached (directly or indirectly) to the inert binding partner; and
a protease cleavage site separating the immune cell engaging domain and the inert binding partner, wherein the protease cleavage site is capable of releasing the inert binding partner and half-life extending moiety from the immune cell engaging domain in the presence of a protease (1) expressed by the cancer or (2) colocalized to the cancer by a targeting moiety that binds a tumor antigen expressed by the cancer and that is the same or different from the targeting moiety in the agent,
wherein cleavage of the protease cleavage site causes loss of the inert binding partner and allows for complementation with the other immune cell engaging domain that is not part of the agent, further wherein if the immune cell engaging domain comprises a VH domain, the other immune cell engaging domain comprises a VL domain and if the immune cell engaging domain comprises a VL domain, the other immune cell engaging domain comprises a VH domain.
This disclosure also describes a component for use in a kit or composition for treating cancer in a patient comprising a first targeted immune cell engaging agent comprising:
an immune cell selection moiety capable of selectively targeting an immune cell;
an immune cell engaging domain capable of immune cell binding activity when binding another immune cell engaging domain, wherein the other immune cell engaging domain is not part of the first component, and wherein the immune cell engaging domain comprises either a VH domain or VL domain, optionally wherein the immune cell engaging domain is a T-cell engaging domain;
an inert binding partner for the immune cell engaging domain binding to the immune cell engaging domain such that the immune cell engaging domain does not bind to the other immune cell engaging domain unless the inert binding partner is removed, wherein if the immune cell engaging domain comprises a VH domain, the inert binding partner comprises a VL domain and if the immune cell engaging domain comprises a VL domain, the inert binding partner comprises a VH domain;
a half-life extending moiety, wherein the half-life extending moiety is attached (directly or indirectly) to the inert binding partner; and
a protease cleavage site separating the immune cell engaging domain and the inert binding partner, wherein the protease cleavage site is capable of releasing the inert binding partner and half-life extending moiety from the immune cell engaging domain in the presence of a protease (1) expressed by the cancer or (2) colocalized to the cancer by a targeting moiety that binds a tumor antigen expressed by the cancer and that is the same or different from the targeting moiety in the agent,
wherein cleavage of the protease cleavage site causes loss of the inert binding partner and allows for complementation with the other immune cell engaging domain that is not part of the agent, further wherein if the immune cell engaging domain comprises a VH domain, the other immune cell engaging domain comprises a VL domain and if the immune cell engaging domain comprises a VL domain, the other immune cell engaging domain comprises a VH domain.
This application also describes an agent for treating cancer in a patient comprising:
a first targeting moiety that binds a tumor antigen expressed by the cancer;
a first T-cell engaging domain capable of T-cell binding activity when binding a second T-cell engaging domain, wherein the first T-cell engaging domain comprises either a VH domain or VL domain;
a second T-cell engaging domain capable of T-cell binding activity when binding a first T-cell engaging domain, wherein the second T-cell engaging domain comprises either a VH domain or VL domain;
a first inert binding partner for the first T-cell engaging domain binding to the first T-cell engaging domain such that the first T-cell engaging domain does not bind to the second T-cell engaging domain unless the inert binding partner is removed, wherein if the first T-cell engaging domain comprises a VH domain, the inert binding partner comprises a VL domain and if the first T-cell engaging domain comprises a VL domain, the inert binding partner comprises a VH domain; and
a protease cleavage site separating the first T-cell engaging domain and the first inert binding partner, wherein the protease cleavage site is capable of releasing the inert binding partner from the T-cell engaging domain in the presence of a protease (1) expressed by the cancer or in the cancer microenvironment; or (2) colocalized to the cancer by a targeting moiety that binds a tumor antigen expressed by the cancer and that is the same or different from the targeting moiety in the agent;
wherein the first and second T-cell engaging domains are capable of binding a T cell when neither is bound to an inert binding partner, and further wherein if the first T-cell engaging domain comprises a VH domain, the second T-cell engaging domain comprises a VL domain and if the first T-cell engaging domain comprises a VL domain, the second T-cell engaging domain comprises a VH domain.
This disclosure also describes an agent for treating cancer in a patient comprising:
an immune cell selection moiety capable of selectively targeting an immune cell;
a first immune cell engaging domain capable of immune cell binding activity when binding a second immune cell engaging domain, wherein the first immune cell engaging domain comprises either a VH domain or VL domain, optionally wherein the first immune cell engaging domain comprises a T-cell engaging domain;
a second immune cell engaging domain capable of immune cell binding activity when binding a first immune cell engaging domain, wherein the second immune cell engaging domain comprises either a VH domain or VL domain, optionally wherein the second immune cell engaging domain comprises a T-cell engaging domain;
a first inert binding partner for the first immune cell engaging domain binding to the first immune cell engaging domain such that the first immune cell engaging domain does not bind to the second immune cell engaging domain unless the inert binding partner is removed, wherein if the first immune cell engaging domain comprises a VH domain, the inert binding partner comprises a VL domain and if the first immune cell engaging domain comprises a VL domain, the inert binding partner comprises a VH domain; and
a protease cleavage site separating the first immune cell engaging domain and the first inert binding partner, wherein the protease cleavage site is capable of releasing the inert binding partner from the immune cell engaging domain in the presence of a protease (1) expressed by the cancer or in the cancer microenvironment; or (2) colocalized to the cancer by a targeting moiety that binds a tumor antigen expressed by the cancer and that is the same or different from the targeting moiety in the agent;
wherein the first and second immune cell engaging domains are capable of binding an immune cell when neither is bound to an inert binding partner, and further wherein if the first immune cell engaging domain comprises a VH domain, the second immune cell engaging domain comprises a VL domain and if the first immune cell engaging domain comprises a VL domain, the second immune cell engaging domain comprises a VH domain.
In some embodiments, an agent further comprises a second targeting moiety that is capable of targeting the cancer.
In some embodiments, an agent further comprises a second inert binding partner for the second immune cell engaging domain binding to the second immune cell engaging domain such that the second immune cell engaging domain does not bind to the first immune cell engaging domain unless the inert binding partner is removed, wherein if the second immune cell or engaging domain comprises a VH domain, the inert binding partner comprises a VL domain and if the second immune cell engaging domain comprises a VL domain, the inert binding partner comprises a VH domain, optionally wherein the second immune cell engaging domain comprises a T-cell engaging domain; and a protease cleavage site separating the second immune cell engaging domain and the second inert binding partner, wherein the protease cleavage site is capable of releasing the inert binding partner from the immune cell engaging domain in the presence of a protease (1) expressed by the cancer or in the cancer microenvironment; or (2) colocalized to the cancer by a targeting moiety that binds a tumor antigen expressed by the cancer and that is the same or different from the targeting moiety in the agent, wherein the first and second immune cell engaging domains are capable of binding an immune cell when neither is bound to an inert binding partner, and further wherein if the first immune cell engaging domain comprises a VH domain, the second immune cell engaging domain comprises a VL domain and if the first immune cell engaging domain comprises a VL domain, the second immune cell engaging domain comprises a VH domain.
In some embodiments, a linker attaches the first and second inert binding partners. In some embodiments, a linker comprises a half-life extending moiety. In some embodiments, a linker is capable of dissociation with the first and/or second inert binding partner upon cleavage of the protease cleavage sites.
In some embodiments, a second component further comprises a second half-life extending moiety, wherein the second half-life extending moiety is attached (directly or indirectly) to the second inert binding partner. In some embodiments, a first and/or second half-life extending moiety is directly attached to the first and/or second inert binding partner. In some embodiments, a first and/or second half-life extending moiety is indirectly attached to the first and/or second inert binding partner via a linker.
In some embodiments, a first component comprises two copies of a first targeting moiety; two copies of a first immune or T-cell engaging domain; and two copies of a first inert binding partner, wherein a protease cleavage site separates both inert binding partners from their respective immune or T-cell engaging domains.
In some embodiments, one end of the half-life extending moiety is attached (directly or indirectly) to one copy of the first inert binding partner and the other end of the half-life extending moiety is attached (directly or indirectly) to the other copy of the first inert binding partner.
In some embodiments, a second component comprises two copies of a second targeting moiety; two copies of a second immune or T-cell engaging domain; and two copies of a second inert binding partner, wherein a protease cleavage sites separates both inert binding partners from their respective immune or T-cell engaging domains.
In some embodiments, one end of the half-life extending moiety is attached (directly or indirectly) to one copy of the second inert binding partner and the other end of the half-life extending moiety is attached (directly or indirectly) to the other copy of the second inert binding partner.
In some embodiments, the two copies of the targeting moiety are the same. In some embodiments, the two copies of the immune or T-cell engaging domain are the same. In some embodiments, the two copies of the inert binding partner are the same. In some embodiments, the two copies of the protease cleavage site separating the inert binding partners from their respective immune or T-cell engaging domains are the same. In some embodiments, the two copies of a protease cleavage site separating the inert binding partners from their respective immune or T-cell engaging domains are different.
In some embodiments, the half-life is decreased after dissociation of one or more half-life extending moieties. In some embodiments, the half-life of the first and/or second component is longer than the half-life of a complex formed by the association of the first and second immune cell or T-cell engaging domains in the form capable of binding to an immune or T cell.
In some embodiments, the first component and/or second component has a half-life greater or equal to 2 days, 4 days, or 7 days. In some embodiments, the agent or component has a half-life greater or equal to 2 days, 4 days, or 7 days.
In some embodiments, the protease cleavage sites are different. In some embodiments, the protease cleavage sites are the same.
In some embodiments, one or more protease cleavage sites are cleaved by a protease expressed by the cancer. In some embodiments, one or more protease cleavage sites are cleaved by a protease that is colocalized to the cancer by a targeting moiety that binds a tumor antigen expressed by the cancer and that is the same or different from the targeting moiety in the agent.
In some embodiments, one or more first and second inert binding partners are capable of dissociation once at least one protease cleavage site for each inert binding partner has been cleaved and after dissociation the two immune cell or T-cell engaging domains that had been bound by the inert binding partners are capable of binding to each other and exhibiting immune cell or T-cell binding activity.
In some embodiments, one or more half-life extending moieties are capable of dissociation together with one or more inert binding partner to which it is attached.
In some embodiments, the one or more half-life extending moieties comprise all or part of an immunoglobulin constant (Fc) domain, serum albumin, serum albumin binding protein, an unstructured protein, and/or PEG. In some embodiments, the one or more half-life extending moieties comprise all or part of an immunoglobulin Fc domain. In some embodiments, the Fc domain comprises the sequence of a human immunoglobulin. In some embodiments, the immunoglobulin is IgG. In some embodiments, the IgG is IgG1, IgG2, or IgG4.
In some embodiments, the Fc domain comprises a naturally occurring sequence.
In some embodiments, the Fc domain comprises one or more mutations as compared to a naturally occurring sequence.
In some embodiments, the Fc domain is an Fc domain with a longer half-life compared to a naturally occurring sequence. In some embodiments, the Fc domain with a longer half-life has increased FcRn binding. In some embodiments, the increased FcRn binding is measured at pH 6.0. In some embodiments, the Fc domain with a longer half-life comprises M252Y/S254T/T256E substitutions. In some embodiments, the Fc domain with a longer half-life comprises M428L/N434S substitutions.
In some embodiments, one or more half-life extending moieties comprise all or part of serum albumin. In some embodiments, the serum albumin is human.
In some embodiments, one or more half-life extending moieties comprise all or part of a serum albumin binding protein. In some embodiments, the serum albumin binding protein is a DARPin, a nanobody, a single-chain variable fragment (scFv), or an antigen-binding fragment (Fab). In some embodiments, the serum albumin binding protein comprises all or part of an albumin binding domain.
In some embodiments, one or more half-life extending moieties comprise all or part of an unstructured protein. In some embodiments, the unstructured protein is an unstructured hydrophilic, biodegradable protein polymer. In some embodiments, the unstructured protein is XTEN.
In some embodiments, one or more half-life extending moieties comprise all or part of PEG.
In some embodiments, the first and second half-life extending moieties are different. In some embodiments, the first and second half-life extending moieties are the same. In some embodiments, the first component is not covalently bound to the second component. In some embodiments, the first component is covalently bound to the second component. In some embodiments, the first component is covalently bound to the second component by a linker comprising a a protease cleavage site.
In some embodiments, the immune cell selection moieties capable of selectively targeting an immune cell selectively targets a T cell, a macrophage, a natural killer cell, a neutrophil, an eosinophil, a basophil, a γδ T cell, a natural killer T cell (NKT cells), or an engineered immune cell.
In some embodiments, the immune cell selection moieties capable of selectively targeting an immune cell selectively targets a T cell, optionally where the T cell is a CD8+ or CD4+ T cell. In some embodiments, the immune cell selection moiety targets CD8, CD4, or CXCR3, or does not specifically bind regulatory T cells. In some embodiments, the immune cell selection moiety comprises an aptamer or an antibody or antigen-specific binding fragment thereof.
In some embodiments, the aptamer or antibody or antigen-specific binding fragment thereof specifically binds an antigen on a T cell. In some embodiments, the first and second T-cell or immune cell engaging domains are capable of binding CD3 or the T cell receptor (TCR) when neither is bound to an inert binding partner. In some embodiments, the first and second T-cell or immune cell engaging domains are capable of forming a Fv when not bound to an inert binding partner.
In some embodiments, the one or more targeting moieties are an antibody or antigen-binding fragment thereof. In some embodiments, the antibody or antigen-binding fragment thereof is (i) specific for any of 4-1BB, 5T4, ACVRL1, ALK1, AXL, B7-H3, BCMA, c-MET, CD133, C4.4a, CA6, CA9, Cadherin-6, CD123, CD133, CD138, CD19, CD20, CD22, CD25, CD27L, CD30, CD33, CD37, CD38, CD44v6, CD56, CD70, CD74(TROP2), CD79b, CEA, CEACAM5, cKit, CLL-1, Cripto, CS1, DLL3, EDNRB, EFNA4, EGFR, EGFRvIII, ENPP3, EpCAM, EPHA2, FGFR2, FGFR3, FLT3, FOLR, FOLR1, GD2, gpA33, GPC3, GPNMB, GUCY2C, HER2, HER3, HLAA2, IGF1-r, IL13RA2, Integrin alpha, LAMP-1, LewisY, LIV-1, LRRC15, MMP9, MSLN, MUC1, MUC16, NaPi2b, Nectin-4, NOTCH3, p-CAD, PD-L1, PSMA, PTK7, ROR1, SLC44A4, SLITRK6, SSTR2, STEAP1, TAG72, TF, TIM-1, or TROP-2, or (ii) an anti-epidermal growth factor receptor antibody; an anti-Her2 antibody; an anti-CD20 antibody; an anti-CD22 antibody; an anti-CD70 antibody; an anti-CD33 antibody; an anti-MUC1 antibody; an anti-CD40 antibody; an anti-CD74 antibody; an anti-P-cadherin antibody; an anti-EpCAM antibody; an anti-CD138 antibody; an anti-E-cadherin antibody; an anti-CEA antibody; an anti-FGFR3 antibody; an anti-mucin core protein antibody; an anti-transferrin antibody; an anti-gp95/97 antibody; an anti-p-glycoprotein antibody; an anti-TRAIL-R1 antibody; an anti-DR5 antibody; an anti-IL-4 antibody; an anti-IL-6 antibody; an anti-CD19 antibody; an anti-PSMA antibody; an anti-PSCA antibody; an anti-Cripto antibody; an anti-PD-L1 antibody; an anti-IGF-1R antibody; an anti-CD38 antibody; an anti-CD133 antibody; an anti-CD123 antibody; an anti-CDE49d antibody; an anti-glypican 3 antibody; an anti-cMET antibody; or an anti-IL-13R antibody.
In some embodiments, the antibody or antigen-binding fragment comprises all or part of the amino acid sequence of 1C1, (GS) 5745, ABBV-085, ABBV-399, ABBV-838, AbGn-107, ABT-414, ADCT-301, ADCT-402, AGS-16C3F, AGS62P1, AGS67E, AMG 172d, AMG 595d, Andecaliximab, Anetumab ravtansine, ARX788, ASG-15MEd, ASG-5MEk, Atezolizumab, AVE1642, AVE9633e, Avelumab, BAY1129980, BAY1187982e, BAY79-4620b, BIIB015d, Bivatuzumab mertansineb, BMS-986148, Brentuximab vedotin, Cantuzumab mertansine, CC49, CDX-014, Cirmtuzumab, Coltuximab ravtansine, DEDN6526Ae, Denintuzumab mafodotin, Depatuxizumab, DFRF4539Ad, DMOT4039Ae, DS-8201A, Durvalumab, Enfortumab vedotin, Farletuzumab, FLYSYN, Gatipotuzumab, Gemtuzumab ozogamicin, Glembatumumab vedotin, GSK2857916, HKT288, Hu3F8, HuMax-AXL-ADC, IDEC-159, IMGN289b, IMGN388a, IMGN529, Indatuximab ravtansine, Inotuzumab ozogamicin, Istiratumab, Labetuzumab govitecan, Lifastuzumab vedotin, LOP628h, Lorvotuzumab mertansine, LY3076226, MCLA-117 (CLEC-12AxCD3), MDX-1203d, MEDI-4276, MEDI-547b, Milatuzumab-doxorubicin, Mirvetuximab soravtansine, MLN0264, MLN2704e, MM-302i, Mosunetuzumab, MOv18 IgE, Ocrelizumab, Oportuzumab, Patritumab, PCA-062, PF-03446962, PF-06263507a, PF-06647020, PF-06647263, PF-06650808d, Pinatuzumab vedotin, Polatuzumab vedotin, PSMA ADC 301c, RC48-ADC, Rituximab, Rovalpituzumab tesirine, Sacituzumab, Sacituzumab govitecan, SAR408701, SAR428926, SAR566658, SC-002, SC-003, SGN-15a, SGN-CD123A, SGN-CD19B, SGN-CD70A, SGN-LIV1A, Sofituzumab vedotin, Solitomab, SSTR2xCD3 XmAb18087, STRO-002, SYD-985, Talacotuzumab, Tisotumab vedotin, Trastuzumab emtansine, U3-1402, Ublituximab, Vadastuximab talirine, Vandortuzumab vedotin, Vorsetuzumab mafodotin, XMT-1522, or Zenocutuzumab.
In some embodiments, one or more targeting moieties are an aptamer. In some embodiments, the aptamer comprises DNA. In some embodiments, the aptamer comprises RNA. In some embodiments, the aptamer is single-stranded. In some embodiments, the aptamer is a target cell-specific aptamer chosen from a random candidate library. In some embodiments, the aptamer is an anti-EGFR aptamer. In some embodiments, the aptamer binds to the antigen on the cancer cell with a Kd from 1 picomolar to 500 nanomolar. In some embodiments, the aptamer binds to the cancer with a Kd from 1 picomolar to 100 nanomolar.
In some embodiments, one or more targeting moieties comprise IL-2, IL-4, IL-6, α-MSH, transferrin, folic acid, EGF, TGF, PD1, IL-13, stem cell factor, insulin-like growth factor (IGF), or CD40. In some embodiments, one or more targeting moiety comprise a full-length sequence of IL-2, IL-4, IL-6, α-MSH, transferrin, folic acid, EGF, TGF, PD1, IL-13, stem cell factor, insulin-like growth factor (IGF), or CD40. In some embodiments, one or more targeting moiety comprise a truncated form, analog, variant, or derivative of IL-2, IL-4, IL-6, α-MSH, transferrin, folic acid, EGF, TGF, PD1, IL-13, stem cell factor, insulin-like growth factor (IGF), or CD40. In some embodiments, one or more targeting moiety bind a target on the cancer comprising IL-2 receptor, IL-4, IL-6, melanocyte stimulating hormone receptor (MSH receptor), transferrin receptor (TR), folate receptor 1 (FOLR), folate hydroxylase (FOLH1), EGF receptor, PD-L1, PD-L2, IL-13R, CXCR4, IGFR, or CD40L.
In some embodiments, the first and second targeting moieties bind the same antigen. In some embodiments, the first and second targeting moieties bind the same epitope.
In some embodiments, the first and second targeting moieties are the same. In some embodiments, the first and second targeting moieties are different.
In some embodiments, the first and second targeting moieties bind different antigens.
In some embodiments, the first and second targeting moieties bind different epitopes of the same antigen (i.e., protein).
This disclosure also describes a method of treating cancer expressing a tumor antigen that binds the first targeting moiety in a patient comprising administering an agent or component to the patient.
In some embodiments, the cancer expressing a tumor antigen that binds the first targeting moiety is any one of breast cancer, ovarian cancer, endometrial cancer, cervical cancer, bladder cancer, renal cancer, melanoma, lung cancer, prostate cancer, testicular cancer, thyroid cancer, brain cancer, esophageal cancer, gastric cancer, pancreatic cancer, colorectal cancer, liver cancer, leukemia, myeloma, nonHodgkin lymphoma, Hodgkin lymphoma, acute myeloid leukemia, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, lymphoproliferative disorder, myelodysplastic disorder, myeloproliferative disease or premalignant disease.
This disclosure also describes a method of targeting an immune response of a patient to cancer comprising administering the agent or component described herein to the patient.
In some embodiments, the T cells express CD3 or TCR and the T cell engaging domain binds CD3 or TCR.
In some embodiments, if the patient has regulatory T cells in the tumor, the selective immune cell engaging agent does not target markers present on regulatory immune cells (including, but not limited to CD4 and CD25).
This disclosure also describes a method of treating cancer expressing a tumor antigen in a patient comprising administering a composition comprising a component described in this disclosure, wherein the first targeting moiety binds the tumor antigen and a second component comprising a half-life extending moiety.
This disclosure also describes a method of treating cancer expressing a tumor antigen in a patient comprising administering a composition comprising a component described in this disclosure, wherein the first targeting moiety binds the tumor antigen and a second component not comprising a half-life extending moiety.
In some embodiments, one or more nucleic acid molecules encodes an agent or component.
Additional objects and advantages will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice. The objects and advantages will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims.
It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the claims.
The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate one (several) embodiment(s) and together with the description, serve to explain the principles described herein.
Table 1A provides a listing of certain sequences referenced herein. Table 1B provides a listing of certain construct sequences used herein.
METDTLLLWVLLLWVPGSTGELVMTQ
METDTLLLWVLLLWVPGSTGQAVVTQ
METDTLLLWVLLLWVPGSTGEVQLVE
METDTLLLWVLLLWVPGSTGDIQMTQ
METDTLLLWVLLLWVPGSTGDKTHTC
METDTLLLWVLLLWVPGSTGDKTHTC
METDTLLLWVLLLWVPGSTGDKTHTC
METDTLLLWVLLLWVPGSTGDKTHTC
METDTLLLWVLLLWVPGSTGDKTHTC
METDTLLLWVLLLWVPGSTGDKTHTC
GGGSGGGGSGGGGSEVQLLEQSGAELV
SQVQLVESGGGVVQPGRSLRLSCAASGF
RGSGGSGGADDIVMTQTPLSLSVTPGQP
GGGSGGGGSGGGGSEVQLLEQSGAELV
SQVQLVESGGGVVQPGRSLRLSCAASGF
R
GSGGSGGADDIVMTQTPLSLSVTPGQP
GSDIVMTQTPLSLSVTPGQPASISCKSSQS
GTSTGSG
AIPVSLR
GSGGSGGADQVQLV
GSDIVMTQTPLSLSVTPGQPASISCKSSQS
GTSTGSG
AIPVSLR
GSGGSGGADQVQLV
GGGSGGGGSGGGGSEVQLLEQSGAELV
SDIVMTQTPLSLSVTPGQPASISCKSSQSL
GTSTGSGGSGGSGGADQVQLVESGGGV
TSTGSG
AIPVSLR
GSGGSGGADQVQLVE
TSTGSG
AIPVSLR
GSGGSGGADQVQLVE
R
GSGGSGGADQVQLVESGGGVVQPGRS
R
GSGGSGGADQVQLVESGGGVVQPGRS
GGSESKYGPPCPPCPAPEFLGGPSVFLFPP
SDIVMTQSPDSLAVSLGERVTMNCKSSQS
SDIVMTQSPDSLAVSLGERVTMNCKSSQS
GGGGSGGGGSDIVMTQTPLSLSVTPGQP
DDDDKGGGGSGGGGSGSGGSGGADQVQ
GGGGSGGGGSDIVMTQTPLSLSVTPGQP
DDDDKGGGGSGGGGSGSGGSGGADQVQ
SGGGSQVQLQQSGAELAKPGASVKMSC
GSESKYGPPCPPCPAPEFLGGPSVFLFPPK
A
GSDIVMTQTPLSLSVTPGQPASISCKSSQS
GTSTGSG
AIPVSLR
GSGGSGGADQVQLV
GGGSGGGGSGGGGSEVQLLEQSGAELV
SQVQLVESGGGVVQPGRSLRLSCAASGF
R
GSGGSGGADDIVMTQTPLSLSVTPGQP
METDTLLLWVLLLWVPGSTGDAHKSE
METDTLLLWVLLLWVPGSTGDAHKSE
GGGGSGGGGSDIVMTQTPLSLSVTPGQP
DDDDKGGGGSGGGGSGSGGSGGADQVQ
Two-component TEACs and ATTACs described in this invention may comprise half-life extending moieties.
Two-component TEACs are described in U.S. Pat. No. 10,035,856, the contents of which are incorporated herein by reference in their entirety. A two-component TEAC comprises two components, wherein at least one component targets a tumor antigen. Exemplary TEACs in U.S. Pat. No. 10,035,086 include SEQ ID NOs: 165-177. FIGS. 1-4C of U.S. Pat. No. 10,035,856 demonstrate how TEACs mediate T-cell activation.
In contrast, two-component ATTACs comprise at least one targeting moiety that binds a tumor antigen and one immune selection moiety capable of selectively targeting an immune cell. The term ATTAC refers to an antibody tumor-targeting assembly complex.
Two-component ATTACs refer to using one ATTAC component that binds to a cancer antigen and one ATTAC component that does not bind to a cancer antigen, but instead selectively targets an immune cell. Thus, the ATTAC components do not have a “parallel” configuration (as in TEACs), but instead have a “trans” configuration.
In two-component TEACs or ATTACs at least one of the components will be bound to an inert binding partner that may be removed by a cleavage at a cleavage site, wherein the cleavage site is:
a. cleaved by an enzyme expressed by the cancer cells;
b. cleaved through a pH-sensitive cleavage reaction inside the cancer cell;
c. cleaved by a complement-dependent cleavage reaction; or
d. cleaved by a protease that is colocalized to the cancer cell by a targeting moiety that is the same or different from the targeting moiety in the agent.
In an ATTAC or TEAC component or pair, a first component, namely a targeted immune cell binding agent, comprises:
i. a targeting moiety capable of targeting the cancer;
ii. a first immune cell engaging domain capable of immune cell engaging activity when binding a second immune cell engaging domain, wherein the second immune cell engaging domain is not part of the first component. If an inert binding partner is on the targeted immune cell binding agent of the first component, it prevents the first immune cell engaging domain from binding to the second immune cell engaging domain unless the inert binding partner is removed.
In a two-component TEAC described herein, a second component comprises a second targeting moiety and a second immune cell engaging domain capable of immune cell engaging activity when binding the first immune cell engaging domain, wherein the first and second immune cell engaging domains are capable of binding when neither is bound to an inert binding partner. If an inert binding partner is on the targeted immune cell binding agent of the second component, it prevents the first immune cell engaging domain from binding to the second immune cell engaging domain unless the inert binding partner is removed.
In a two-component ATTAC described herein, a second component comprises an immune cell selection moiety capable of selectively targeting an immune cell and a second immune cell engaging domain capable of immune cell engaging activity when binding the first immune cell engaging domain, wherein the first and second immune cell engaging domains are capable of binding when neither is bound to an inert binding partner. If an inert binding partner is on second component in an ATTAC, it prevents the first immune cell engaging domain from binding to the second immune cell engaging domain unless the inert binding partner is removed.
Thus, both components of a two-component TEAC described herein may bind a tumor antigen expressed by the cancer. In contrast, one component of a two-component ATTAC described herein may bind a tumor antigen expressed by the cancer, while the other component selectively targets an immune cell. In this way, a component that binds a tumor antigen expressed by the cancer may be one component of a two-component TEAC or ATTAC, and the agent is either a TEAC or ATTAC based on the nature of the second component.
The moieties described below may be comprised in the ATTACs and TEACs described herein.
A. Half-Life Extending Moieties
A “half-life extending moiety,” as used herein, refers to a moiety that increases the in vivo half-life of an agent or component. A number of different methodologies have been described for increasing the in vivo half-life of biologic therapies (see Wang et al., Protein Cell 9(1):63-73 (2018) and Strohl BioDrugs 29:215-239 (2015)); however, the location of a half-life extending moiety in this context can impact its function and relationship to the composition and therapeutic strategy for treating a patient.
In some embodiments, a first component of a two-component TEAC or ATTAC comprises a half-life extending moiety.
In some embodiments, a half-life extending moiety is attached (directly or indirectly) to an inert binding partner. By “attached directly,” it is meant that there are no amino acids between a half-life extending moiety and the inert binding partner. By “attached indirectly,” it is meant that there are additional amino acids between a half-life extending moiety and the inert binding partner. In some embodiments, a half-life extending moiety is indirectly attached to the first and/or second inert binding partner via a linker.
In some embodiments, the second component of a two-component TEAC or ATTAC further comprises a second half-life extending moiety, wherein the second half-life extending moiety is attached (directly or indirectly) to the second inert binding partner. In some embodiments, the first and second half-life extending moieties are the same. In some embodiments, the first and second half-life extending moieties are different.
In some embodiments, one end of the half-life extending moiety is attached (directly or indirectly) to one copy of the first inert binding partner and the other end of the half-life extending moiety is attached (directly or indirectly) to the other copy of the first inert binding partner.
In some embodiments, one or more half-life extending moieties are capable of dissociation together with one or more inert binding partner to which it is attached.
In some embodiments, the half-life of the agent is decreased after dissociation of one or more half-life extending moieties. In some embodiments, the half-life of the first and/or second component is longer than the half-life of a complex formed by the association of the first and second immune cell or T-cell engaging domains in the form capable of binding to an immune or T cell.
In some embodiments, the first component and/or second component has a half-life greater or equal to 2 days, 4 days, or 7 days. In some embodiments, the agent or component has a half-life greater or equal to 2 days, 4 days, or 7 days.
In some embodiments, the half-life extending moiety is attached to one or more inert binding partners. In some embodiments, when a cleavage site is cleaved, the inert binding partner that is released from the agent/component dissociates together with the half-life extending moiety.
In some embodiments, dissociation of the half-life extending moiety together with the inert binding partner reduces the half-life of the agent or component. In this way, the half-life of an “activated” agent or component (i.e., an agent or component following cleavage to release the inert binding partner and half-life extending moiety) are relatively short, while the half-life of the agent or component prior to cleavage is longer based on the presence of the half-life extending moiety.
In this way, the relatively short half-life of an agent or component in the post-cleavage phase is maintained. This reduces the risk of over-activation of the immune system by active post-cleaved agents or components with long half-lives.
In contrast, the half-life of an agent or component before cleavage is longer based on the presence of a half-life extending moiety. In some embodiments, a longer half-life allows longer time periods between administration of doses of the agent or component. In some embodiments, a longer half-life allows lower doses of an agent or component to be administered.
In a two-component TEAC or ATTAC, both components may comprise a half-life extending moiety. In this way, both components of a two-component TEAC or ATTAC have a longer half-life based on the presence of a half-life extending moiety in each component. In some embodiments, both half-life extending moieties are the same. In some embodiments, the half-life extension moieties are different.
In some embodiments, one or more half-life extending moieties comprise all or part of an immunoglobulin constant (Fc) domain, serum albumin, serum albumin binding protein, an unstructured protein, and/or PEG.
1. Fc Domains
In some embodiments, the one or more half-life extending moieties comprises all or part of an Fc domain. In some embodiments, the Fc domain comprises the sequence of a human immunoglobulin. In some embodiments, the immunoglobulin is IgG. In some embodiments, the IgG is IgG1, IgG2, or IgG4.
In some embodiments, one or both component of a two-component TEAC or ATTAC may comprise an Fc domain as a half-life extending moiety. When a single component of a two-component TEAC or ATTAC comprises an Fc domain, it may be referred to as an “IgG TEAC” or “IgG ATTAC.” When both components of a two-component TEAC or ATTAC comprise an Fc domain, the two components may be referred to as “Dual IgG TEACs,” or “Dual IgG ATTACs.” Using the term Dual refers to having two separate components, each utilizing an IgG such as the two constructs in
In some embodiments, a TEAC component comprising a CH domain can pair with an identical TEAC component in a cell based on pairing of the CH domains.
In some embodiments, the Fc domain comprises a naturally occurring sequence.
In some embodiments, the Fc domain comprises one or more mutations as compared to a naturally occurring sequence. An Fc domain comprising one or more mutation in an Fc domain may be referred to as an engineered Fc domain. A variety of engineered Fc domains have been described that may comprised in a TEAC or ATTAC (see Wang et al., Protein Cell 9(1):63-73 (2018)).
In some embodiments, the Fc domain is an IgG4/IgG1 hybrid or an IgG2 with IgG1 hinge.
In some embodiments, the Fc domain is an Fc domain with a longer half-life compared to a naturally occurring sequence (See Robbie et al., Antimicrob Agents Chemother 57(12):6147-6153 (2013) and Zalevsky et al., Nat Biotechnol 28(2):157-159 (2010)). In some embodiments, the Fc domain with a longer half-life has increased FcRn binding. In some embodiments, the increased FcRn binding is measured at pH 6.0. In some embodiments, the Fc domain with a longer half-life comprises M252Y/S254T/T256E substitutions. In some embodiments, the Fc domain with a longer half-life comprises M428L/N434S substitutions.
2. Serum Albumin or Albumin-Binding Protein
In some embodiments, one or more half-life extending moiety comprises all or part of serum albumin. In some embodiments, the serum albumin is human. SEQ ID NOs: 210-211 represent exemplary TEACs comprising human serum albumin.
In some embodiments, one or more half-life extending moiety comprises all or part of a serum albumin binding protein. In some embodiments, the serum albumin binding protein is a DARPin, a nanobody, a single-chain variable fragment (scFv), or an antigen-binding fragment (Fab). In some embodiments, the serum albumin binding protein comprises all or part of an albumin binding domain.
For example, half-life extension using serum albumin-binding DARPin domains has been described in Steiner et al., Protein Engineering, Design & Selection 30(9):583-591 (2017). Similarly, nanobodies that bind serum albumin have been shown to increase the half-life of linked nanobodies (see Hoefman et al., Antibodies 4:141-156 (2015)).
3. Unstructured Proteins or PEG
In some embodiments, one or more half-life extending moiety comprises all or part of an unstructured protein. In some embodiments, the unstructured protein is an unstructured hydrophilic, biodegradable protein polymer. In some embodiments, the unstructured protein is XTEN.
In some embodiments, one or more half-life extending moiety comprises all or part of polyethylene glycol (PEG).
B. Targeting Moiety Capable of Targeting the Cancer
Targeting moieties for TEACs have been described in U.S. Pat. No. 10,035,856, the contents of which are incorporated herein by reference in their entirety, including non-antibody binding partners and aptamers that may be comprised as targeting moieties capable of targeting cancer (such as SEQ ID NOs: 95-164).
The first component as a two-component ATTAC can comprise the same targeting moieties that can be comprised in a TEAC.
The targeting moiety functions in the ATTAC or TEAC to deliver the agent to the local environment of unwanted cells, enabling a localized treatment strategy. In some embodiments, the unwanted cells are cancer cells. In certain embodiments, the targeting moiety targets the cancer cells by specifically binding to the cancer cells.
In some embodiments comprising two targeting moieties, the first and second targeting moieties bind the same antigen. In some embodiments comprising two targeting moieties, the first and second targeting moieties bind the same epitope. In some embodiments comprising two targeting moieties, the first and second targeting moieties are the same. In some embodiments comprising two targeting moieties, the first and second targeting moieties are different. In some embodiments comprising two targeting moieties, the first and second targeting moieties bind different antigens. In some embodiments comprising two targeting moieties, the first and second targeting moieties bind different epitopes of the same antigen (i.e., protein).
In some embodiments, the targeting moiety is an antibody or antigen-binding fragment thereof. By antigen-binding fragment, we mean any antibody fragment that retains its binding activity to the target on the cancer cell, such as an scFv or other functional fragment including an immunoglobulin devoid of light chains, VHH, VNAR, Fab, Fab′, F(ab′)2, Fv, antibody fragment, diabody, scAB, single-domain heavy chain antibody, single-domain light chain antibody, Fd, CDR regions, or any portion or peptide sequence of the antibody that is capable of binding antigen or epitope. VHH and VNAR are alternatives to classical antibodies and even though they are produced in different species (camelids and sharks, respectively), we will also include them in antigen-binding fragments of antibodies. Unless specifically noted as “full length antibody,” when the application refers to antibody it inherently includes a reference to an antigen-binding fragment thereof.
Table 2A provide additional information about cancers that may be targeting with different targeting moieties, including the fact that some targeting moieties may be able to target a number of different types of cancer.
Antibodies that have bind tumor antigens and that have specificity for tumor cells are well-known in the art. Table 2B summarizes selected publications on exemplary antibodies that bind tumor antigens and that could be used as targeting moieties in the invention.
II of her2 and acidic variants thereof). This application discloses the variable
CH3)2 antibody fragments (minibodies) for tumor targeting, Protein Eng
region of tumor associated EGFR, Proc Natl Acad Sci USA 106(13): 5082-
antibodies). This patent discloses CDR sequences in its FIG. 1.
membrane antigen (PSMA) lacking in fucosyl residues). This application
antibody drug conjugates). FIGS. 1-4 of this application show heavy and
methods of use thereof). This application discloses CDR sequences in its
antigen). This application discloses CDR sequences in its FIGS. 26-29.
c-Met). This application discloses CDRs in its FIGS. 1-3 and heavy and
receptors). This application discloses VH and VL sequences in its claims 1-2.
apoptosis-inducing ligand receptor and uses thereof). This application
and uses thereof). This application discloses CDR sequence data in
Antibody Fragments to Human Vascular Endothelial Growth Factor-C Block
Its Interaction with VEGF Receptor-2 and 3, PLoS One 5(8): e11941 (2010).
proteins). This application discloses VH and VL sequences in its claim 17.
antibodies for cancer targeting and detection). This application discloses
Peptide Selected from Phage Display Libraries Recognize Unique Epitopes
and Predominantly Bind Adenocarcinoma, Cancer Res. 58(19): 4324-32
MUC5AC overexpression, J Immunol 182(4): 2349-56 (2009) at page 3,
human scFv antibody against CEA protein, BMC Cancer 6: 41 (2006). This
24p4c12 Proteins). This application discloses light and heavy chain variable
Proteins). This application discloses light and heavy chain variable domain
proteins). This application iscloses light and was heavy chain variable domain
24P4C12 proteins). This application discloses light and heavy chain variable
24P4C12 proteins). This application discloses light and heavy chain variable
thereof). This application discloses light and heavy chain variable domain
neuropilin-antibody complexes). This application discloses light and heavy
chain). This application discloses the heavy chain variable region in its claim
antibody). This application discloses the heavy chain sequence in its claim 6
use thereof). This application discloses CDR sequences in its
interactions with human T lymphocytes). This application discloses that anti-
monoclonal antibodies and therapeutic agents). This application discloses
thereof). This application discloses antibody sequence in its claims 22-23.
Inhibition of Fgfr3). This application discloses scFv sequences in claim 6
fibroblast growth factor receptor 3 with human single-chain Fv
antibodies inhibits bladder carcinoma cell line proliferation. Clin Cancer Res
Drug Dev Technol. 8(1): 27-36. (2010).
Ther. 4(3): 369-79 (2005).
Mol Cancer Ther. 8(9): 2616-2624 (2009).
Cancer 45(14): 2579-87 (2009) (abstract).
J Thorac Oncol. 3: 1379-1383 (2008).
J Coll Physicians Surg Pak 16(2): 148-9 (2006) (abstract).
Natl Acad Sci 19(19): 12293-12297 (2002).
Immunol Lett 84(1): 57-62 (2002) (abstract).
Natl Acad Sci 101(29): 10691-10696 (2004).
Blood 112(3): 711-720 (2008).
J Haematol 127: 404-415 (2004) (abstract).
Res 17(20): 6448-6458 (2011).
Hemat 64: 275-281 (2000) (abstract).
Blood 92: 4066-4071 (1998).
Cancer Sci 101(1): 224-30 (2010) (epub 2009 Sep. 8) (abstract).
The FDA maintains listings of approved antibody drugs for treating cancer, many of which bind to cancer antigens and can be employed in this context. See The Orange Book Online or Drugs@FDA on the FDA website. The FDA also maintains listings of clinical trials in progress in the clinicaltrials.gov database, which may be searched by disease names. Table 2C provides a representative list of approved antibodies with specificity for tumor cells. Table 2D provides a representative list of antibodies in development with specificity for tumor cells.
Other antibodies well-known in the art may be used as targeting moieties to target to a given cancer. The antibodies and their respective antigens include nivolumab (anti-PD-1 Ab), TA99 (anti-gp75), 3F8 (anti-GD2), 8H9 (anti-B7-H3), abagovomab (anti-CA-125 (imitation)), adecatumumab (anti-EpCAM), afutuzumab (anti-CD20), alacizumab pegol (anti-VEGFR2), altumomab pentetate (anti-CEA), amatuximab (anti-mesothelin), AME-133 (anti-CD20), anatumomab mafenatox (anti-TAG-72), apolizumab (anti-HLA-DR), arcitumomab (anti-CEA), bavituximab (anti-phosphatidylserine), bectumomab (anti-CD22), belimumab (anti-BAFF), besilesomab (anti-CEA-related antigen), bevacizumab (anti-VEGF-A), bivatuzumab mertansine (anti-CD44 v6), blinatumomab (anti-CD19), BMS-663513 (anti-CD137), brentuximab vedotin (anti-CD30 (TNFRSF8)), cantuzumab mertansine (anti-mucin CanAg), cantuzumab ravtansine (anti-MUC1), capromab pendetide (anti-prostatic carcinoma cells), carlumab (anti-MCP-1), catumaxomab (anti-EpCAM, CD3), cBR96-doxorubicin immunoconjugate (anti-Lewis-Y antigen), CC49 (anti-TAG-72), cedelizumab (anti-CD4), Ch. 14.18 (anti-GD2), ch-TNT (anti-DNA associated antigens), citatuzumab bogatox (anti-EpCAM), cixutumumab (anti-IGF-1 receptor), clivatuzumab tetraxetan (anti-MUC1), conatumumab (anti-TRAIL-R2), CP-870893 (anti-CD40), dacetuzumab (anti-CD40), daclizumab (anti-CD25), dalotuzumab (anti-insulin-like growth factor I receptor), daratumumab (anti-CD38 (cyclic ADP ribose hydrolase)), demcizumab (anti-DLL4), detumomab (anti-B-lymphoma cell), drozitumab (anti-DR5), duligotumab (anti-HER3), dusigitumab (anti-ILGF2), ecromeximab (anti-GD3 ganglioside), edrecolomab (anti-EpCAM), elotuzumab (anti-SLAMF7), elsilimomab (anti-IL-6), enavatuzumab (anti-TWEAK receptor), enoticumab (anti-DLL4), ensituximab (anti-SAC), epitumomab cituxetan (anti-episialin), epratuzumab (anti-CD22), ertumaxomab (anti-HER2/neu, CD3), etaracizumab (anti-integrin αvβ3), faralimomab (anti-Interferon receptor), farletuzumab (anti-folate receptor 1), FBTA05 (anti-CD20), ficlatuzumab (anti-HGF), figitumumab (anti-IGF-1 receptor), flanvotumab (anti-TYRP1 (glycoprotein 75)), fresolimumab (anti-TGF (3), futuximab (anti-EGFR), galiximab (anti-CD80), ganitumab (anti-IGF-I), gemtuzumab ozogamicin (anti-CD33), girentuximab (anti-carbonic anhydrase 9 (CAIX)), glembatumumab vedotin (anti-GPNMB), guselkumab (anti-IL13), ibalizumab (anti-CD4), ibritumomab tiuxetan (anti-CD20), icrucumab (anti-VEGFR-1), igovomab (anti-CA-125), IMAB362 (anti-CLDN18.2), IMC-CS4 (anti-CSF1R), IMC-TR1 (TGFβRII), imgatuzumab (anti-EGFR), inclacumab (anti-selectin P), indatuximab ravtansine (anti-SDC1), inotuzumab ozogamicin (anti-CD22), intetumumab (anti-CD51), ipilimumab (anti-CD152), iratumumab (anti-CD30 (TNFRSF8)), KM3065 (anti-CD20), KW-0761 (anti-CD194), LY2875358 (anti-MET) labetuzumab (anti-CEA), lambrolizumab (anti-PDCD1), lexatumumab (anti-TRAIL-R2), lintuzumab (anti-CD33), lirilumab (anti-KIR2D), lorvotuzumab mertansine (anti-CD56), lucatumumab (anti-CD40), lumiliximab (anti-CD23 (IgE receptor)), mapatumumab (anti-TRAIL-R1), margetuximab (anti-ch4D5), matuzumab (anti-EGFR), mavrilimumab (anti-GMCSF receptor a-chain), milatuzumab (anti-CD74), minretumomab (anti-TAG-72), mitumomab (anti-GD3 ganglioside), mogamulizumab (anti-CCR4), moxetumomab pasudotox (anti-CD22), nacolomab tafenatox (anti-C242 antigen), naptumomab estafenatox (anti-5T4), narnatumab (anti-RON), necitumumab (anti-EGFR), nesvacumab (anti-angiopoietin 2), nimotuzumab (anti-EGFR), nivolumab (anti-IgG4), nofetumomab merpentan, ocrelizumab (anti-CD20), ocaratuzumab (anti-CD20), olaratumab (anti-PDGF-R a), onartuzumab (anti-c-MET), ontuxizumab (anti-TEM1), oportuzumab monatox (anti-EpCAM), oregovomab (anti-CA-125), otlertuzumab (anti-CD37), pankomab (anti-tumor specific glycosylation of MUC1), parsatuzumab (anti-EGFL7), pascolizumab (anti-IL-4), patritumab (anti-HER3), pemtumomab (anti-MUC1), pertuzumab (anti-HER2/neu), pidilizumab (anti-PD-1), pinatuzumab vedotin (anti-CD22), pintumomab (anti-adenocarcinoma antigen), polatuzumab vedotin (anti-CD79B), pritumumab (anti-vimentin), PRO131921 (anti-CD20), quilizumab (anti-IGHE), racotumomab (anti-N-glycolylneuraminic acid), radretumab (anti-fibronectin extra domain-B), ramucirumab (anti-VEGFR2), rilotumumab (anti-HGF), robatumumab (anti-IGF-1 receptor), roledumab (anti-RHD), rovelizumab (anti-CD11 & CD18), samalizumab (anti-CD200), satumomab pendetide (anti-TAG-72), seribantumab (anti-ERBB3), SGN-CD19A (anti-CD19), SGN-CD33A (anti-CD33), sibrotuzumab (anti-FAP), siltuximab (anti-IL-6), solitomab (anti-EpCAM), sontuzumab (anti-episialin), tabalumab (anti-BAFF), tacatuzumab tetraxetan (anti-alpha-fetoprotein), taplitumomab paptox (anti-CD19), telimomab aritox, tenatumomab (anti-tenascin C), teneliximab (anti-CD40), teprotumumab (anti-CD221), TGN1412 (anti-CD28), ticilimumab (anti-CTLA-4), tigatuzumab (anti-TRAIL-R2), TNX-650 (anti-IL-13), tositumomab (anti-CS20), tovetumab (anti-CD140a), TRBS07 (anti-GD2), tregalizumab (anti-CD4), tremelimumab (anti-CTLA-4), TRU-016 (anti-CD37), tucotuzumab celmoleukin (anti-EpCAM), ublituximab (anti-CD20), urelumab (anti-4-1BB), vantictumab (anti-Frizzled receptor), vapaliximab (anti-AOC3 (VAP-1)), vatelizumab (anti-ITGA2), veltuzumab (anti-CD20), vesencumab (anti-NRP1), visilizumab (anti-CD3), volociximab (anti-integrin α5β1), vorsetuzumab mafodotin (anti-CD70), votumumab (anti-tumor antigen CTAA16.88), zalutumumab (anti-EGFR), zanolimumab (anti-CD4), zatuximab (anti-HER1), ziralimumab (anti-CD147 (basigin)), RG7636 (anti-ETBR), RG7458 (anti-MUC16), RG7599 (anti-NaPi2b), MPDL3280A (anti-PD-L1), RG7450 (anti-STEAP1), and GDC-0199 (anti-Bcl-2).
Antibodies that bind these antigens may also be used as targeting moieties, especially for the types of cancers noted: aminopeptidase N (CD13), annexin A1, B7-H3 (CD276, various cancers), CA125 (ovarian cancers), CA15-3 (carcinomas), CA19-9 (carcinomas), L6 (carcinomas), Lewis Y (carcinomas), Lewis X (carcinomas), alpha fetoprotein (carcinomas), CA242 (colorectal cancers), placental alkaline phosphatase (carcinomas), prostate s7pecific antigen (prostate), prostatic acid phosphatase (prostate), epidermal growth factor (carcinomas), CD2 (Hodgkin's disease, NHL lymphoma, multiple myeloma), CD3 epsilon (T-cell lymphoma, lung, breast, gastric, ovarian cancers, autoimmune diseases, malignant ascites), CD19 (B cell malignancies), CD20 (non-Hodgkin's lymphoma, B-cell neoplasmas, autoimmune diseases), CD21 (B-cell lymphoma), CD22 (leukemia, lymphoma, multiple myeloma, SLE), CD30 (Hodgkin's lymphoma), CD33 (leukemia, autoimmune diseases), CD38 (multiple myeloma), CD40 (lymphoma, multiple myeloma, leukemia (CLL)), CD51 (metastatic melanoma, sarcoma), CD52 (leukemia), CD56 (small cell lung cancers, ovarian cancer, Merkel cell carcinoma, and the liquid tumor, multiple myeloma), CD66e (carcinomas), CD70 (metastatic renal cell carcinoma and non-Hodgkin lymphoma), CD74 (multiple myeloma), CD80 (lymphoma), CD98 (carcinomas), CD123 (leukemia), mucin (carcinomas), CD221 (solid tumors), CD22 (breast, ovarian cancers), CD262 (NSCLC and other cancers), CD309 (ovarian cancers), CD326 (solid tumors), CEACAM3 (colorectal, gastric cancers), CEACAM5 (CEA, CD66e) (breast, colorectal and lung cancers), DLL4 (A-like-4), EGFR (various cancers), CTLA4 (melanoma), CXCR4 (CD 184, heme-oncology, solid tumors), Endoglin (CD 105, solid tumors), EPCAM (epithelial cell adhesion molecule, bladder, head, neck, colon, NHL prostate, and ovarian cancers), ERBB2 (lung, breast, prostate cancers), FCGR1 (autoimmune diseases), FOLR (folate receptor, ovarian cancers), FGFR (carcinomas), GD2 ganglioside (carcinomas), G-28 (a cell surface antigen glycolipid, melanoma), GD3 idiotype (carcinomas), heat shock proteins (carcinomas), HER1 (lung, stomach cancers), HER2 (breast, lung and ovarian cancers), HLA-DR10 (NHL), HLA-DRB (NHL, B cell leukemia), human chorionic gonadotropin (carcinomas), IGF1R (solid tumors, blood cancers), IL-2 receptor (T-cell leukemia and lymphomas), IL-6R (multiple myeloma, RA, Castleman's disease, IL6 dependent tumors), integrins (αvβ3, α5β1, α6β4, α11β3, α5β5, αvβ5, for various cancers), MAGE-1 (carcinomas), MAGE-2 (carcinomas), MAGE-3 (carcinomas), MAGE 4 (carcinomas), anti-transferrin receptor (carcinomas), p97 (melanoma), MS4A1 (membrane-spanning 4-domains subfamily A member 1, Non-Hodgkin's B cell lymphoma, leukemia), MUC1 (breast, ovarian, cervix, bronchus and gastrointestinal cancer), MUC16 (CA125) (ovarian cancers), CEA (colorectal cancer), gp100 (melanoma), MARTI (melanoma), MPG (melanoma), MS4A1 (membrane-spanning 4-domains subfamily A, small cell lung cancers, NHL), nucleolin, Neu oncogene product (carcinomas), P21 (carcinomas), nectin-4 (carcinomas), paratope of anti-(N-glycolylneuraminic acid, breast, melanoma cancers), PLAP-like testicular alkaline phosphatase (ovarian, testicular cancers), PSMA (prostate tumors), PSA (prostate), ROB04, TAG 72 (tumour associated glycoprotein 72, AML, gastric, colorectal, ovarian cancers), T-cell transmembrane protein (cancers), Tie (CD202b), tissue factor, TNFRSF10B (tumor necrosis factor receptor superfamily member 10B, carcinomas), TNFRSF13B (tumor necrosis factor receptor superfamily member 13B, multiple myeloma, NHL, other cancers, RA and SLE), TPBG (trophoblast glycoprotein, renal cell carcinoma), TRAIL-R1 (tumor necrosis apoptosis inducing ligand receptor 1, lymphoma, NHL, colorectal, lung cancers), VCAM-1 (CD106, Melanoma), VEGF, VEGF-A, VEGF-2 (CD309) (various cancers). Some other tumor associated antigen targets have been reviewed (Gerber, et al, mAbs 2009 1:247-253; Novellino et al, Cancer Immunol Immunother. 2005 54:187-207, Franke, et al, Cancer Biother Radiopharm. 2000, 15:459-76, Guo, et al., Adv Cancer Res. 2013; 119: 421-475, Parmiani et al. J Immunol. 2007 178:1975-9). Examples of these antigens include Cluster of Differentiations (CD4, CDS5, CD6, CD7, CD8, CD9, CD10, CD11a, CD11b, CD11c, CD12w, CD14, CD15, CD16, CDw17, CD18, CD21, CD23, CD24, CD25, CD26, CD27, CD28, CD29, CD31, CD32, CD34, CD35, CD36, CD37, CD41, CD42, CD43, CD44, CD45, CD46, CD47, CD48, CD49b, CD49c, CD53, CD54, CD55, CD58, CD59, CD61, CD62E, CD62L, CD62P, CD63, CD68, CD69, CD71, CD72, CD79, CD81, CD82, CD83, CD86, CD87, CD88, CD89, CD90, CD91, CD95, CD96, CD100, CD103, CD105, CD106, CD109, CD117, CD120, CD127, CD133, CD134, CD135, CD138, CD141, CD142, CD143, CD144, CD147, CD151, CD152, CD154, CD156, CD158, CD163, CD166, CD168, CD184, CDw186, CD195, CD202 (a, b), CD209, CD235a, CD271, CD303, CD304), annexin A1, nucleolin, endoglin (CD105), ROB04, amino-peptidase N, -like-4 (DLL4), VEGFR-2 (CD309), CXCR4 (CD184), Tie2, B7-H3, WT1, MUC1, LMP2, HPV E6 E7, EGFRvIII, HER-2/neu, idiotype, MAGE A3, p53 nonmutant, NY-ESO-1, GD2, CEA, MelanA/MART1, Ras mutant, gp100, p53 mutant, proteinase3 (PR1), bcr-abl, tyrosinase, survivin, hTERT, sarcoma translocation breakpoints, EphA2, PAP, ML-IAP, AFP, EpCAM, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, ALK, androgen receptor, cyclin B 1, polysialic acid, MYCN, RhoC, TRP-2, GD3, fucosyl GM1, mesothelin, PSCA, MAGE A1, sLe(a), CYPIB I, PLAC1, GM3, BORIS, Tn, GloboH, ETV6-AML, NY-BR-1, RGS5, SART3, STn, carbonic anhydrase IX, PAXS, OY-TES1, sperm protein 17, LCK, HMWMAA, AKAP-4, SSX2, XAGE 1, B7H3, legumain, Tie 2, Page4, VEGFR2, MAD-CT-1, FAP, PDGFR-β, MAD-CT-2, and Fos-related antigen 1.
In some embodiments, the antibody or antigen-binding fragment thereof is specific for 4-1BB, 5T4, ACVRL1, ALK1, AXL, B7-H3, BCMA, c-MET, CD133, C4.4a, CA6, CA9, Cadherin-6, CD123, CD133, CD138, CD19, CD20, CD22, CD25, CD27L, CD30, CD33, CD37, CD38, CD44v6, CD56, CD70, CD74(TROP2), CD79b, CEA, CEACAM5, cKit, CLL-1, Cripto, CS1, DLL3, EDNRB, EFNA4, EGFR, EGFRvIII, ENPP3, EpCAM, EPHA2, FGFR2, FGFR3, FLT3, FOLR, FOLR1, GD2, gpA33, GPC3, GPNMB, GUCY2C, HER2, HER3, HLAA2, IGF1-r, IL13RA2, Integrin alpha, LAMP-1, LewisY, LIV-1, LRRC15, MMP9, MSLN, MUC1, MUC16, NaPi2b, Nectin-4, NOTCH3, p-CAD, PD-L1, PSMA, PTK7, ROR1, SLC44A4, SLITRK6, SSTR2, STEAP1, TAG72, TF, TIM-1, or TROP-2.
In some embodiments, the antibody or antigen-binding fragment thereof is specific for Her2/Neu, CD22, PSMA, CD30, CD2, CD33, CD8, CD86, CD, CA125, Carbonic Anhydrase IX, CD70, CD74, CD56, CD40, CD19, e-met/HGFR, TRAIL-R1, DRS, PD-1, IGF-1R, VEGF-R2, PSCA, MUC1, CanAg, Mesothelin, P-cadherin, Myostatin, Cripto, ACVRL 1/ALK1, MUCSAC, CEACAM, CD137, CXCR4, Neuropilin 1, Glypicans, HERS/EGFR, PDGFRa, EphA2, CD38, CD138/Syndecanl, A4-integrin, EpCAM, ADAM17, CD59, Integrin aVf33, MCP-1, PCLA, RANKL, RG1, SLC44A4, STEAP-1, VEGF-C, CCN1, CD44, CD98, c-RET, DLL4, Episialin, GPNMB, Integrin α6β4, LFL2, LIV-1, Ly6E, MUC18, NRP1, Phosphatidylserine, PRLR, TACSTD-2, Tenascin C, TWEAKR, VANGL2, PD-L1, PD-L2, BCMA, DKK-1, ICAM-q, GRP78, FGFR3, SLAMF6, CD48, CD71, APRIL, DR5, CD37, HLA-DR, CD70b, CAIX, TPBG, ENPP3, FGFR1, VEGFR-2, CLDN18, GCC, C242, FGFR2, GPR49, IGFR, ALK, GC2, EGFRvIII, CD4, CD5, IL-3Ra, Integrin α5β1, Lewis y/b antigen, EGFL7, NaPi2b, flt4, CD133, CD123, CD45, c-Kit, Lewis Y, Siglec-15, FLT-3, CEACAM1, Cadherin-19, GM3, TYRP1, GD3, MUC5A, CLDN6, Glypican-3, FGFR4, PIVKA-II, PLVAP, Progastrin, CEA, CLDN1, A33, CK8, or FAP.
In some embodiments, the antibody or antigen-binding fragment thereof is an anti-epidermal growth factor receptor antibody; an anti-Her2 antibody; an anti-CD20 antibody; an anti-CD22 antibody; an anti-CD70 antibody; an anti-CD33 antibody; an anti-MUC1 antibody; an anti-CD40 antibody; an anti-CD74 antibody; an anti-P-cadherin antibody; an anti-EpCAM antibody; an anti-CD138 antibody; an anti-E-cadherin antibody; an anti-CEA antibody; an anti-FGFR3 antibody; an anti-mucin core protein antibody; an anti-transferrin antibody; an anti-gp95/97 antibody; an anti-p-glycoprotein antibody; an anti-TRAIL-R1 antibody; an anti-DR5 antibody; an anti-IL-4 antibody; an anti-IL-6 antibody; an anti-CD19 antibody; an anti-PSMA antibody; an anti-PSCA antibody; an anti-Cripto antibody; an anti-PD-L1 antibody; an anti-IGF-1R antibody; an anti-CD38 antibody; an anti-CD133 antibody; an anti-CD123 antibody; an anti-CDE49d antibody; an anti-glypican 3 antibody; an anti-cMET antibody; or an anti-IL-13R antibody.
In some embodiments, the antibody or antigen-binding fragments comprises all or part of the amino acid sequence of 1C1, (GS) 5745, ABBV-085, ABBV-399, ABBV-838, AbGn-107, ABT-414, ADCT-301, ADCT-402, AGS-16C3F, AGS62P1, AGS67E, AMG 172d, AMG 595d, Andecaliximab, Anetumab ravtansine, ARX788, ASG-15MEd, ASG-5MEk, Atezolizumab, AVE1642, AVE9633e, Avelumab, BAY1129980, BAY1187982e, BAY79-4620b, BIIB015d, Bivatuzumab mertansineb, BMS-986148, Brentuximab vedotin, Cantuzumab mertansine, CC49, CDX-014, Cirmtuzumab, Coltuximab ravtansine, DEDN6526Ae, Denintuzumab mafodotin, Depatuxizumab, DFRF4539Ad, DMOT4039Ae, DS-8201A, Durvalumab, Enfortumab vedotin, Farletuzumab, FLYSYN, Gatipotuzumab, Gemtuzumab ozogamicin, Glembatumumab vedotin, GSK2857916, HKT288, Hu3F8, HuMax-AXL-ADC, IDEC-159, IMGN289b, IMGN388a, IMGN529, Indatuximab ravtansine, Inotuzumab ozogamicin, Istiratumab, Labetuzumab govitecan, Lifastuzumab vedotin, LOP628h, Lorvotuzumab mertansine, LY3076226, MCLA-117 (CLEC-12AxCD3), MDX-1203d, MEDI-4276, MEDI-547b, Milatuzumab-doxorubicin, Mirvetuximab soravtansine, MLN0264, MLN2704e, MM-302i, Mosunetuzumab, MOv18 IgE, Ocrelizumab, Oportuzumab, Patritumab, PCA-062, PF-03446962, PF-06263507a, PF-06647020, PF-06647263, PF-06650808d, Pinatuzumab vedotin, Polatuzumab vedotin, PSMA ADC 301c, RC48-ADC, Rituximab, Rovalpituzumab tesirine, Sacituzumab, Sacituzumab govitecan, SAR408701, SAR428926, SAR566658, SC-002, SC-003, SGN-15a, SGN-CD123A, SGN-CD19B, SGN-CD70A, SGN-LIV1A, Sofituzumab vedotin, Solitomab, SSTR2xCD3 XmAb18087, STRO-002, SYD-985, Talacotuzumab, Tisotumab vedotin, Trastuzumab emtansine, U3-1402, Ublituximab, Vadastuximab talirine, Vandortuzumab vedotin, Vorsetuzumab mafodotin, XMT-1522, or Zenocutuzumab.
In some embodiments, the targeting moiety capable of targeting a cancer is not an antibody, but is another type of targeting moiety. A wide range of targeting moieties capable of targeting cancer are known, including DNA aptamers, RNA aptamers, albumins, lipocalins, fibronectins, ankyrins, CH1/2/3 scaffolds (including abdurins (IgG CH2 scaffolds)), fynomers, Obodies, DARPins, knotins, avimers, atrimers, anticallins, affilins, affibodies, bicyclic peptides, cys-knots, FN3 (adnectins, centryrins, pronectins, TN3), and Kunitz domains. These and other non-antibody scaffold structures may be used for targeting to a cancer cell. Smaller non-antibody scaffolds are rapidly removed from the bloodstream and have a shorter half-life than monocolonal antibodies. They also show faster tissue penetration owing to fast extravasation from the capillary lumen through the vascular endothelium and basement membrane. See Vazquez-Lombardi et al., Drug Discovery Today 20(1):1271-1283 (2015). A number of non-antibody scaffolds targeting cancer are already under clinical development, with other candidates in the preclinical stage, as shown in Table 2E. See Vazquez-Lombardi, Table 1.
In some embodiments, one or more targeting moiety comprises IL-2, IL-4, IL-6, α-MSH, transferrin, folic acid, EGF, TGF, PD1, IL-13, stem cell factor, insulin-like growth factor (IGF), or CD40. In some embodiments, one or more targeting moiety comprises a full-length sequence of IL-2, IL-4, IL-6, α-MSH, transferrin, folic acid, EGF, TGF, PD1, IL-13, stem cell factor, insulin-like growth factor (IGF), or CD40. In some embodiments, one or more targeting moiety comprises a truncated form, analog, variant, or derivative of IL-2, IL-4, IL-6, α-MSH, transferrin, folic acid, EGF, TGF, PD1, IL-13, stem cell factor, insulin-like growth factor (IGF), or CD40. In some embodiments, one or more targeting moiety binds a target on the cancer comprising IL-2 receptor, IL-4, IL-6, melanocyte stimulating hormone receptor (MSH receptor), transferrin receptor (TR), folate receptor 1 (FOLR), folate hydroxylase (FOLH1), EGF receptor, PD-L1, PD-L2, IL-13R, CXCR4, IGFR, or CD40L.
In some embodiments, one or more targeting moiety is an aptamer. In some embodiments, the aptamer comprises DNA. In some embodiments, the aptamer comprises RNA. In some embodiments, the aptamer is single-stranded.
In some embodiments, the aptamer is a target cell-specific aptamer chosen from a random candidate library. In some embodiments, the aptamer is an anti-EGFR aptamer. In some embodiments, the aptamer binds to the antigen on the cancer cell with a Kd from 1 picomolar to 500 nanomolar. In some embodiments, the aptamer binds to the cancer with a Kd from 1 picomolar to 100 nanomolar.
Additional specific targeting moieties include those provided in Table 3.
C. Immune Cell Selection Moiety
In some embodiments, an ATTAC comprises a targeting moiety and an immune cell selection moiety. In other words, an ATTAC may comprise one component that binds to an antigen on a cancer cell and another component that binds to an immune cell.
In some embodiments, an ATTAC comprises an immune cell selection moiety specific for a particular immune cell. In some embodiments, the immune cell selection moiety is specific for CD8+ T cells, CD4+ T cells, natural killer (NK) cells, macrophages, neutrophils, eosinophils, basophils, γδ T cells, natural killer T cells (NKT cells), or engineered immune cells. Engineered immune cells refers to immune cells with engineered receptors with new specificity. Examples of engineered immune cells include chimeric antigen receptor (CAR) T cells, NK, NKT, or γδ T cells.
In some embodiments, the immune cell selection moiety targets an immune cell marker that is not a tumor antigen. In some embodiments, the immune cell selection moiety allows targeting of an ATTAC to an immune cell, wherein the immune cell is not a cancer cell. In some embodiments, the immune cell selection moiety does not target the ATTAC to a lymphoma, myeloma, or leukemia. In some embodiments, the ATTAC targets a solid tumor (in other words any tumor not of an immune cell).
In some embodiments, the immune cell selection moiety does not specifically bind regulatory T cells. In some embodiments, the immune cell selection moiety does not specifically bind TH17 cells. In some embodiments, the selective immune cell binding agent does not target markers present on regulatory immune cells (including, but not limited to CD4 and CD25).
Table 4 lists some representative immune cell selection moieties for different desired immune cells.
Escherichia coli. J Microbiol Biotechnol. 21(5): 529-
D. Immune Cell Engaging Domain
The immune cell engaging domain functions are capable of immune cell engaging activity when a first immune cell engaging domain binds to a second immune cell engaging domain. When the first and second immune cell engaging domains are paired together, when the inert binding partner is removed, they can bind to an immune cell. This binding can lead to activation of the immune cell.
In the absence of pairing of the first and second immune cell engaging domain, neither the first nor the second immune cell engaging domain alone can bind to an immune cell.
In some embodiments, the first and second immune cell engaging domains are capable of forming an Fv when not bound to an inert binding partner.
In some embodiments, the immune cell is a T cell, natural killer cell, macrophage, neutrophil, eosinophil, basophil, γδ T cell, NKT cell, or engineered immune cell. In some embodiments, the first and second immune cell engaging domains when paired together can activate an immune cell.
An ATTAC can engage a range of immune cells. In some embodiments, a TEAC or ATTAC engages a T cell.
1. T-Cell Engaging Domains
In some embodiments, the immune cell engaging domain is a T-cell engaging domain. The targeted T-cell engaging agent comprises a first T-cell engaging domain that is unable of engaging a T-cell alone. Instead, the first T-cell engaging domain is capable of activity when binding a second T-cell engaging domain, which is not part of the targeted T-cell engaging agent. Thus, the first and second T-cell engaging domains may be any two moieties that do not possess T-cell engaging activity alone, but do possess it when paired with each other. In other words, the first and second T-cell engaging domains are complementary halves of a functional active protein.
In some embodiments, the first and second T-cell or immune cell engaging domains are capable of forming a Fv when not bound to an inert binding partner
In some embodiments, the first and second T-cell or immune cell engaging domains are capable of binding CD3 or the T cell receptor (TCR) when neither is bound to an inert binding partner. When the two T-cell engaging domains are associated together in the two-component system, they may bind to the CD3 antigen and/or T-cell receptor on the surface of the T-cell as these activate T cells. CD3 is present on all T cells and consists of subunits designated γ, δ, ε, ζ, and η. The cytoplasmic tail of CD3 is sufficient to transduce the signals necessary for T cell activation in the absence of the other components of the TCR receptor complex. Normally, activation of T cell cytotoxicity depends first on binding of the TCR with a major histocompatibility complex (MHC) protein, itself bound to a foreign antigen, located on a separate cell. In a normal situation, only when this initial TCR-MHC binding has taken place can the CD3 dependent signally cascade responsible for T cell clonal expansion and, ultimately, T cell cytotoxicity ensue. In some of the present embodiments, however, when the two-component system binds to CD3 and/or the TCR, activation of cytotoxic T cells in the absence of independent TCR-MHC can take place by virtue of the crosslinking of the CD3 and/or TCR molecules mimicking an immune synapse formation. This means that T cells may be cytotoxically activated in a clonally independent fashion, i.e. in a manner that is independent of the specific TCR clone carried by the T cell. This allows for activation of the entire T cell compartment rather than only specific T cells of a certain clonal identity.
In some embodiments, the first T-cell engaging domain comprises a VH domain and the second T-cell engaging domain comprises a VL domain. In other embodiments, the first T-cell engaging domain comprises a VL domain and the second T-cell engaging domain comprises a VH domain. In such embodiments, when paired together the first and second T-cell engaging domains may comprise an scFv (by this we mean equivalent to an scFv but for the fact that the VH and VL are not in a single-chain configuration).
If the first and second T-cell engaging domains are a pair of VH and VL domains, the VH and VL domains may be specific for an antigen expressed on the surface of a T cell, such as CD3 or TCR. If the antigen is CD3, one potential T-cell engaging domain may be derived from muromonab (muromonab-CD3 or OKT3), otelixizumab, teplizumab, visilizumab, foralumab, or SP34. One skilled in the art would be aware of a wide range of anti-CD3 antibodies, some of which are approved therapies or have been clinically tested in human patients (see Kuhn and Weiner Immunotherapy 8(8):889-906 (2016)). Table 5 presents selected publications on exemplary anti-CD3 antibodies.
Diabetes. 54(6): 1763-9 (2005).
Clin Immunol. 149(3): 268-78 (2013) (abstract).
Expert Opin Biol Ther. 10(3): 459-65 (2010).
Clin Immunol. 149(3): 268-78 (2013) (abstract).
Antibodies with specificity to the TCR, including the αβ and γδ TCRs, are also well-known. Table 6 presents selected publications on exemplary anti-TCR antibodies.
J Immunol. 184: 2156-2165 (2010).
J Immunol. 171(7): 3394-400 (2003).
2. Natural Killer Cell Engaging Domains
In some embodiments, the immune cell engaging domain is a natural killer cell engaging domain. When the two natural killer cell engaging domains are associated together in the two-component system, they may bind to an antigen on the surface of the NK cell to engage these cells. In some embodiments, the antigen on the surface of the NK cell may be NKG2D, CD16, NKp30, NKp44, NKp46 or DNAM.
In some embodiments, having one half of the two-component system bind to a surface protein on the natural killer cell and having the other half of the system bind to cancer cells allows specific engagement of natural killer cells. Engagement of natural killer cells can lead to their activation and induce natural killer cell-mediated cytotoxicity and cytokine release.
When the two natural killer cell engaging domains are associated together in the ATTAC, the natural killer cell may specifically lyse the cancer cells bound by the cancer-specific ATTAC component. Killing of a cancer cell may be mediated by either the perforin/granzyme system or by FasL-Fas engagement. As well as this potential cytotoxic function, natural killer cells are also able to secrete proinflammatory cytokines including interferon gamma and tumor necrosis factor alpha which can activate macrophages and dendritic cells in the immediate vicinity to enhance the anti-cancer immune response.
In some embodiments, the first natural killer cell engaging domain comprises a VH domain and the second natural killer cell engaging domain comprises a VL domain. In other embodiments, the first natural killer cell engaging domain comprises a VL domain and the second natural killer cell engaging domain comprises a VH domain. In such embodiments, when paired together the first and second natural killer cell engaging domains may comprise an scFv (by this we mean equivalent to an scFv but for the fact that the VH and VL are not in a single-chain configuration).
If the first and second natural killer cell engaging domains are a pair of VH and VL domains, the VH and VL domains may be specific for an antigen expressed on the surface of a natural killer cell, such as NKG2D, CD16, NKp30, NKp44, NKp46 and DNAM.
Table 7 presents selected publications on some exemplary antibodies specific for an antigen expressed on the surface of a natural killer cell.
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J Biol Chem. 279(52): 53907-14 (2004).
Cancer Res. 64(13): 4664-9 (2004).
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World J Hepatol. 9(25): 1073-1080 (2017).
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Monoclon Antib Immunodiagn Immunother.
World J Hepatol. 9(25): 1073-1080 (2017).
Front Immunol. 7: 413 (2016).
3. Macrophage Engaging Domains
In some embodiments, the immune cell engaging domain is a macrophage engaging domain. As used herein, a “macrophage” may refer to any cell of the mononuclear phagocytic system, such as grouped lineage-committed bone marrow precursors, circulating monocytes, resident macrophages, and dendritic cells (DC). Examples of resident macrophages can include Kupffer cells and microglia.
When the two macrophage engaging domains are associated together in the two-component system, they may bind to an antigen on the surface of the macrophage to engage these cells. In some embodiments, the antigen on the surface of the macrophage may be CD89 (Fc alpha receptor 1), CD64 (Fc gamma receptor 1), CD32 (Fc gamma receptor 2A) or CD16a (Fc gamma receptor 3A).
In some embodiments, having one half of the two-component system bind to a surface protein on the macrophage and having the other half of the system bind to cancer cells allows specific engagement of macrophages. Engagement of macrophages can lead the macrophage to phagocytose the cancer cell.
In some embodiments, inducing macrophage phagocytosis via binding to an antigen on the surface of the macrophages is independent of Fc receptor binding, which has been shown previously to be a method of tumor cell killing by macrophages. Normally, cancer cells are bound by whole antibodies and the Fc portion of the antibody binds to the Fc receptor and induces phagocytosis.
In some embodiments, engagement of toll-like receptors on the macrophage surface (see patent application US20150125397A1) leads to engagement of macrophages.
When the two macrophage engaging domains are associated together in the ATTAC, they may induce the macrophage to phagocytose the cancer cell bound by the cancer-specific ATTAC component.
In some embodiments, the first macrophage engaging domain comprises a VH domain and the second macrophage engaging domain comprises a VL domain. In other embodiments, the first macrophage engaging domain comprises a VL domain and the second macrophage engaging domain comprises a VH domain. In such embodiments, when paired together the first and second macrophage engaging domains may comprise an scFv (by this we mean equivalent to an scFv but for the fact that the VH and VL are not in a single-chain configuration).
If the first and second macrophage engaging domains are a pair of VH and VL domains, the VH and VL domains may be specific for an antigen expressed on the surface of a macrophage, such as CD89 (Fc alpha receptor 1), CD64 (Fc gamma receptor 1), CD32 (Fc gamma receptor 2A) and CD16a (Fc gamma receptor 3A), or toll-like receptors.
Table 8 presents selected publications on some exemplary antibodies specific for an antigen expressed on the surface of a macrophage.
J Immunol. 167(5): 2651-6 (2001).
Clin Exp Immunol. 103(1): 161-6 (1996).
Int J Cancer. 135(6): 1497-508 (2014).
PLoS One. 10(9): e0137474 (2015).
J Exp Med. 168(3): 1193-8 (1988).
Ann Rheum Dis. 50(5): 311-315 (1991).
Clin Exp Immunol. 137(3): 529-37 (2004).
Cytometry B Clin Cytom. 82(2): 61-6 (2012).
4. Neutrophil Engaging Domains
In some embodiments, the immune cell engaging domain is a neutrophil engaging domain. When the two neutrophil engaging domains are associated together in the two-component system, they may bind to an antigen on the surface of the neutrophil to engage these cells. In some embodiments, the antigen on the surface of the neutrophil may be CD89 (FcαR1), FcγRI (CD64), FcγRIIA (CD32), FcγRIIIA (CD16a), CD11b (CR3, αMβ2), TLR2, TLR4, CLEC7A (Dectin1), formyl peptide receptor 1 (FPR1), formyl peptide receptor 2 (FPR2), or formyl peptide receptor 3 (FPR3).
In some embodiments, having one half of the two-component system bind to a surface protein on the neutrophil and having the other half of the system bind to cancer cells allows specific engagement of neutrophils. Engagement of neutrophils can lead to phagocytosis and cell uptake.
When the two neutrophil engaging domains are associated together in the ATTAC, the neutrophil may engulf the target cells.
In some embodiments, the first neutrophil engaging domain comprises a VH domain and the second neutrophil engaging domain comprises a VL domain. In other embodiments, the first neutrophil engaging domain comprises a VL domain and the second neutrophil engaging domain comprises a VH domain. In such embodiments, when paired together the first and second neutrophil engaging domains may comprise an scFv (by this we mean equivalent to an scFv but for the fact that the VH and VL are not in a single-chain configuration).
If the first and second neutrophil engaging domains are a pair of VH and VL domains, the VH and VL domains may be specific for an antigen expressed on the surface of a neutrophil, such as CD89 (FcαR1), FcγRI (CD64), FcγRIIA (CD32), FcγRIIIA (CD16a), CD11b (CR3, αMβ2), TLR2, TLR4, CLEC7A (Dectin1), FPR1, FPR2, or FPR3.
Table 9 presents selected publications on some exemplary antibodies specific for an antigen expressed on the surface of a neutrophil.
Journal of Biological Chemistry 292(10):
Cancer Immunol Immunother. 50(2):
Front Immunol. 5: 461 (2014)
Front Immunol. 5: 461 (2014)
5. Eosinophil Engaging Domains
In some embodiments, the immune cell engaging domain is an eosinophil engaging domain. When the two eosinophil engaging domains are associated together in the two-component system, they may bind to an antigen on the surface of the eosinophil to engage these cells. In some embodiments, the antigen on the surface of the eosinophil may be CD89 (Fc alpha receptor 1), FcεRI, FcγRI (CD64), FcγRIIA (CD32), FcγRIIIB (CD16b), or TLR4.
In some embodiments, having one half of the two-component system bind to a surface protein on the eosinophil and having the other half of the system bind to cancer cells allows specific engagement of eosinophils. Engagement of eosinophils can lead to degranulation and release of preformed cationic proteins, such as EPO, major basic protein 1 (MBP1), and eosinophil-associated ribonucleases (EARs), known as ECP and eosinophil-derived neurotoxin.
When the two neutrophil engaging domains are associated together in the ATTAC, the neutrophil may phagocytose the target cell or secrete neutrophil extracellular traps (NETs); finally, they may activate their respiratory burst cascade to kill phagocytosed cells.
In some embodiments, the first eosinophil engaging domain comprises a VH domain and the second eosinophil engaging domain comprises a VL domain. In other embodiments, the first eosinophil engaging domain comprises a VL domain and the second eosinophil engaging domain comprises a VH domain. In such embodiments, when paired together the first and second eosinophil engaging domains may comprise an scFv (by this we mean equivalent to an scFv but for the fact that the VH and VL are not in a single-chain configuration).
If the first and second eosinophil engaging domains are a pair of VH and VL domains, the VH and VL domains may be specific for an antigen expressed on the surface of an eosinophil, such as CD89 (Fc alpha receptor 1), FcεRI, FcγRI (CD64), FcγRIIA (CD32), FcγRIIIB (CD16b), or TLR4.
Table 10 presents selected publications on some exemplary antibodies specific for an antigen expressed on the surface of an eosinophil.
Arch Immunol Ther Exp (Warsz). 49(3): 217-29 (2001)
J Allergy Clin Immunol. 125(2 Suppl 2): S73-80 (2010)
Curr Opin Immunol. 19(2): 239-45 (2007)
Curr Opin Immunol. 19(2): 239-45 (2007)
Curr Opin Immunol. 19(2): 239-45 (2007)
6. Basophil Engaging Domains
In some embodiments, the immune cell engaging domain is a basophil engaging domain. When the two basophil engaging domains are associated together in the two-component system, they may bind to an antigen on the surface of the basophil to engage these cells. In some embodiments, the antigen on the surface of the basophil may be CD89 (Fc alpha receptor 1) or FcεRI.
In some embodiments, having one half of the two-component system bind to a surface protein on the basophil and having the other half of the system bind to cancer cells allows specific engagement of basophils. Engagement of basophils can lead to the release of basophil granule components such as histamine, proteoglycans, and proteolytic enzymes. They also secrete leukotrienes (LTD-4) and cytokines.
When the two basophil engaging domains are associated together in the ATTAC, the basophil may degranulate.
In some embodiments, the first basophil engaging domain comprises a VH domain and the second basophil engaging domain comprises a VL domain. In other embodiments, the first basophil engaging domain comprises a VL domain and the second basophil engaging domain comprises a VH domain. In such embodiments, when paired together the first and second basophil engaging domains may comprise an scFv (by this we mean equivalent to an scFv but for the fact that the VH and VL are not in a single-chain configuration).
If the first and second basophil engaging domains are a pair of VH and VL domains, the VH and VL domains may be specific for an antigen expressed on the surface of a basophil, such as CD89 (Fc alpha receptor 1) or FcεRI.
Table 11 presents selected publications on some exemplary antibodies specific for an antigen expressed on the surface of a basophil.
Arch Immunol Ther Exp (Warsz). 49(3): 217-29 (2001)
J Allergy Clin Immunol. 125(2 Suppl 2): S73-80 (2010)
7. γδ T Cells
In some embodiments, the immune cell engaging domain is a γδ T-cell engaging domain. As used herein, a γδ T cell refers to a T cell having a TCR made up of one gamma chain (γ) and one delta chain (δ).
When the two γδ T-cell engaging domains are associated together in the two-component system, they may bind to an antigen on the surface of the γδ T cell to engage these cells. In some embodiments, the antigen on the surface of the γδ T cell may be γδ TCR, NKG2D, CD3 Complex (CD3ε, CD3γ, CD3δ, CD3ζ, CD3η), 4-1BB, DNAM-1, or TLRs (e.g., TLR2, TLR6).
In some embodiments, having one half of the two-component system bind to a surface protein on the γδ T cell and having the other half of the system bind to cancer cells allows specific engagement of γδ T cells. Engagement of γδ T cell can lead to cytolysis of the target cell and release of proinflammatory cytokines such as TNFα and IFNγ.
When the two γδ T-cell engaging domains are associated together in the ATTAC, the γδ T cell may kill the target cell.
In some embodiments, the first γδ T-cell engaging domain comprises a VH domain and the second γδ T-cell engaging domain comprises a VL domain. In other embodiments, the first γδ T-cell engaging domain comprises a VL domain and the second γδ T-cell engaging domain comprises a VH domain. In such embodiments, when paired together the first and second γδ T-cell engaging domains may comprise an scFv (by this we mean equivalent to an scFv but for the fact that the VH and VL are not in a single-chain configuration).
If the first and second γδ T-cell engaging domains are a pair of VH and VL domains, the VH and VL domains may be specific for an antigen expressed on the surface of a γδ T cell, such as γδ TCR, NKG2D, CD3 Complex (CD3ε, CD3γ, CD3δ, CD3ζ, CD3η), 4-1BB, DNAM-1, or TLRs (TLR2, TLR6).
Table 12 presents selected publications on some exemplary antibodies specific for an antigen expressed on the surface of a γδ T cell.
Eur J Immunol. 39(5): 1361-8 (2009)
8. Natural Killer T Cells (NKT Cells)
In some embodiments, the immune cell engaging domain is a NKT engaging domain. NKT cells refers to T cells that express the Vα24 and Vβ11 TCR receptors.
When the two NKT engaging domains are associated together in the two-component system, they may bind to an antigen on the surface of the NKT to engage these cells. In some embodiments, the antigen on the surface of the NKT may be αβTCR, NKG2D, CD3 Complex (CD3ε, CD3γ, CD3δ, CD3ζ, CD3η), 4-1BB, or IL-12R.
In some embodiments, having one half of the two-component system bind to a surface protein on the NKT and having the other half of the system bind to cancer cells allows specific engagement of NKT. Engagement of NKTs can lead to cytolysis of the target cell.
When the two NKT engaging domains are associated together in the ATTAC, the NKT may cytolysis of the target cell and the release of proinflammatory cytokines.
In some embodiments, the first NKT engaging domain comprises a VH domain and the second NKT engaging domain comprises a VL domain. In other embodiments, the first NKT engaging domain comprises a VL domain and the second NKT engaging domain comprises a VH domain. In such embodiments, when paired together the first and second NKT engaging domains may comprise an scFv (by this we mean equivalent to an scFv but for the fact that the VH and VL are not in a single-chain configuration).
If the first and second NKT engaging domains are a pair of VH and VL domains, the VH and VL domains may be specific for an antigen expressed on the surface of a NKT, such as aβTCR, NKG2D, CD3 Complex (CD3ε, CD3γ, CD3δ, CD3ζ, CD3η), 4-1BB, or IL-12R.
Table 13 presents selected publications on some exemplary antibodies specific for an antigen expressed on the surface of a NKT.
Annu. Rev. Immunol. 16: 523-544 (1998)
Eur J Cancer. 54: 112-119 (2016)
9. Engineered Immune Cells
In some embodiments, the immune cell engaging domain is an engineered immune cell engaging domain.
In some embodiments, the engineered immune cell is a chimeric antigen receptor (CAR) cell. In some embodiments, the CAR comprises an extracellular domain capable of tightly binding to a tumor antigen (for example, an scFv), fused to a signaling domain partly derived from a receptor naturally expressed by an immune cell. Exemplary CARs are described in Facts about Chimeric Antigen Receptor (CAR) T-Cell Therapy, Leukemia and Lymphoma Society, December 2017. CARs may comprise an scFV region specific for a tumor antigen, an intracellular co-stimulatory domain, and linker and transmembrane region. For example, a CAR in a CAR T cell may comprise an extracellular domain of a tumor antigen fused to a signaling domain partly derived from the T cell receptor. A CAR may also comprise a co-stimulatory domain, such as CD28, 4-1 BB, or OX40. In some embodiments, binding of the CAR expressed by an immune cell to a tumor target antigen results in immune cell activation, proliferation, and target cell elimination. Thus, a range of CARs may be used that differ in their scFV region, intracellular co-stimulatory domains, and linker and transmembrane regions to generate engineered immune cells.
Exemplary engineered immune cells include CAR T cells, NK cells, NKT cells, and γδ cells. In some embodiments, engineered immune cells are derived from the patient's own immune cells. In some embodiments, the patient's tumor expresses a tumor antigen that binds to the scFV of the CAR.
Potential CAR targets studied so far include CD19, CD20, CD22, CD30, CD33, CD123, ROR1, Igk light chain, BCMA, LNGFR, and NKG2D. However, the CAR technology would be available for developing engineered immune cells to a range of tumor antigens.
In some embodiments, the engineered immune cell is a genetically engineered immune cell.
When the two engineered immune cell engaging domains are associated together in the two-component system, they may bind to an antigen on the surface of the engineered immune cell to engage these cells. In some embodiments, the antigen on the surface of the engineered immune cell may be an engagement domain recited in this application with specificity for T cells, NK cells, NKT cells, or γδ cells.
In some embodiments, having one half of the two-component system bind to a surface protein on the engineered immune cell and having the other half of the system bind to cancer cells allows specific engagement of engineered immune cells. Engagement of engineered immune cells can lead to activation of the effector response of these cells such as cytolysis of their target and release of cytokines.
When the two engineered immune cell engaging domains are associated together in the ATTAC, the engineered immune cell may kill the target cell.
In some embodiments, the first engineered immune cell engaging domain comprises a VH domain and the second engineered immune cell engaging domain comprises a VL domain. In other embodiments, the first engineered immune cell engaging domain comprises a VL domain and the second engineered immune cell engaging domain comprises a VH domain. In such embodiments, when paired together the first and second engineered immune cell engaging domains may comprise an scFv (by this we mean equivalent to an scFv but for the fact that the VH and VL are not in a single-chain configuration).
If the first and second engineered immune cell engaging domains are a pair of VH and VL domains, the VH and VL domains may be specific for an antigen expressed on the surface of an engineered immune cell, based on the type of cell used for the engineering.
E. Inert Binding Partners
Inert binding partners are described in U.S. Pat. No. 10,035,856. A TEAC or ATTAC can comprise at least one inert binding partner capable of binding the first immune cell or T-cell engaging domain and preventing it from binding to a second immune cell or T-cell engaging domain unless certain conditions occur. In some embodiments, a first and second immune cell engaging domain, such as a T-cell engaging domain, can pair together when neither is bound to an inert binding partner.
In some embodiments, one or more first and second inert binding partners are capable of dissociation once at least one protease cleavage site for each inert binding partner has been cleaved and after dissociation the two immune cell or T-cell engaging domains that had been bound by the inert binding partners are capable of binding to each other and exhibiting immune cell or T-cell binding activity.
In some embodiments, cleavage of one or more protease cleavage site causes dissociation of one or more inert binding partner and complementation of the first and second immune cell or T-cell engaging domain. By complementation, it is meant that the first and second immune cell or T-cell engaging domain can bind to or pair with each together. In some embodiments, the paired (i.e., complemented) first and second immune cell or T-cell engaging domains can bind an antigen on an immune cell, such as a T cell, when neither immune cell or T-cell engaging domain is bound to an inert binding partner.
1. Inactivated VII or VL Domains as Inert Binding Partners
In some embodiments when an immune cell engaging domain comprises a VH or VL domain, the inert binding partner has homology to a corresponding VL or VH domain that can pair with the immune cell engaging domain to form a functional antibody and bind to an immune cell antigen. This immune cell antigen may be an antigen present on any immune cell, including a T cell, a macrophage, a natural killer cell, a neutrophil, eosinophil, basophil, γδ T cell, natural killer T cell (NKT cells), or engineered immune cell. In some embodiments, this immune cell antigen is CD3.
In some embodiments, the inert binding partner comprises a VH or VL that cannot specifically bind an antigen when paired with its corresponding VL or VH of the immune cell engaging domain because of one or more mutations made in the inert binding partner to inhibit binding to the target antigen. In some embodiments, the VH or VL of the inert binding partner may differ by one or more amino acids from a VH or VL specific for an immune cell antigen. In other words, one or more mutations may be made to a VH or VL specific for a target immune cell antigen to generate an inert binding partner.
These mutations may be, for example, a substitution, insertion, or deletion in the polypeptide sequence of a VH or VL specific for an immune cell antigen to generate an inert binding partner. In some embodiments, the mutation in a VH or VL specific for an immune cell antigen may be made within CDR1, CDR2, or CDR3 to generate an inert binding partner. In some embodiments, an VH or VL used as an inert binding partner may retain the ability to pair with an immune cell engaging domain, but the resulting paired VH/VL domains have reduced binding to the immune cell antigen. In some embodiments, an inert binding partner has normal affinity to bind its corresponding immune cell engaging domain, but the paired VH/VL has lower binding affinity for the immune cell antigen compared to a paired VH/VL that does not comprise the mutation of the inert binding partner. For example, this lower affinity may be a 20-fold, 100-fold, or 1000-fold lower binding to an immune cell antigen.
In some embodiments, the first immune cell engaging domain comprises a VH specific for an immune cell antigen and the inert binding partner comprises a VL domain for the same antigen that has one or more mutations such that the paired VH/VL has decreased or no binding to the antigen. In some embodiments, the first immune cell engaging domain comprises a VL specific for an immune cell antigen and the inert binding partner comprises a VH domain for the same antigen that has one or more mutations such that the paired VH/VL has decreased or no binding to the antigen.
In some embodiments, the second immune cell engaging domain comprises a VH specific for an immune cell antigen and the inert binding partner comprises a VL domain for the same antigen that has one or more mutations such that the paired VH/VL has decreased or no binding to the antigen. In some embodiments, the second immune cell engaging domain comprises a VL specific for an immune cell antigen and the inert binding partner comprises a VH domain for the same antigen that has one or more mutations such that the paired VH/VL has decreased or no binding to the antigen.
F. Inert Binding Partners Obtained from Unrelated Antibodies
In some embodiments, a VH or VL used as an inert binding partner is unrelated to the VL or VH of the immune cell engaging domain. In other words, the inert binding partner may have little or no sequence homology to the corresponding VH or VL that normally associates with the VL or VH of the immune cell engaging domain. In some embodiments, the VH or VL used as an inert binding partner may be from a different antibody or scFv than the VL or VH used as the immune cell engaging domain.
If both components have inert binding partner, in some embodiments, the VH inert binding partner of one component and the VL inert binding partner of the other component may be from different antibodies
G. Cleavage Site
A range of different cleavage sites can be used in TEACs and ATTACs. In some embodiments, wherein the cleavage site is cleaved by an enzyme expressed by the cancer cells; cleaved through a pH-sensitive cleavage reaction inside the cancer cell; cleaved by a complement-dependent cleavage reaction; or cleaved by a protease that is colocalized to the cancer cell by a targeting moiety that is the same or different from the targeting moiety in the agent. Cleavage sites are described in U.S. Pat. No. 10,035,856.
In some embodiments, at least one cleavage site may be cleaved by an enzyme expressed by the cancer or in the cancer microenvironment. As used herein, an enzyme (such as a protease) expressed “in the cancer microenvironment” refers to an enzyme that is localized to the vicinity of a cancer cell, but outside the cancer cell. Tumors depend on complex interactions between cancer cells and the surrounding stromal compartment. For example, interactions between cancer cells and its surrounding stroma can promote tumor progression by mechanisms such as remodeling of the extracellular matrix to enhance invasion, which may rely on proteases expressed by stromal cells. In some embodiments, cells in the cancer microenvironment have an altered phenotype that helps to promote tumor survival, growth, or metastasis, such as by increased or altered expression of proteases. In some embodiments, a protease in the cancer microenvironment is a protease expressed by a stromal cell in the vicinity of the cancer. In some embodiments, a protease is secreted by a stromal cell in the vicinity of the cancer. In some embodiments, a protease expressed in the cancer microenvironment is not expressed by the cancer cells themselves.
In addition, cancer cells are known to express certain enzymes, such as proteases, and these may be employed in this strategy to cleave the targeted T-cell engaging agent's cleavage site. In some embodiments, one or more protease cleavage sites are cleaved by a protease expressed by the cancer. By way of nonlimiting example, cathepsin B cleaves FR, FK, VA and VR amongst others; cathepsin D cleaves PRSFFRLGK (SEQ ID NO: 45), ADAM28 cleaves KPAKFFRL (SEQ ID NO: 1), DPAKFFRL (SEQ ID NO: 2), KPMKFFRL (SEQ ID NO: 3) and LPAKFFRL (SEQ ID NO: 4); and MMP2 cleaves AIPVSLR (SEQ ID NO: 46), SLPLGLWAPNFN (SEQ ID NO: 47), HPVGLLAR (SEQ ID NO: 48), GPLGVRGK (SEQ ID NO: 49) (also cleaved by MMP9), and GPLGLWAQ (SEQ ID NO: 50), for example. Other cleavage sites listed in Table 1A may also be employed. Protease cleavage sites and proteases associated with cancer are well known in the art. Oncomine (www.oncomine.org) is an online cancer gene expression database, so when the agent of the invention is for treating cancer, the skilled person may search the Oncomine database to identify a particular protease cleavage site (or two protease cleavage sites) that will be appropriate for treating a given cancer type. Alternative databases include the European Bioinformatic Institute (www.ebi.ac.uk), in particular (www.ebi.ac.uk/gxa). Protease databases include PMAP (www.proteolysis.org), ExPASy Peptide Cutter (ca.expasy.org/tools/peptidecutter) and PMAP.Cut DB (cutdb.burnham.org).
In some embodiments, at least one cleavage site may be cleaved through a pH-sensitive cleavage reaction inside the cancer cell. If the ATTAC or TEAC is internalized into the cell, the cleavage reaction may occur inside the cell and may be triggered by a change in pH between the microenvironment outside the unwanted cell and the interior of the cell. Specifically, some cancer types are known to have acidic environments in the interior of the cancer cells. Such an approach may be employed when the interior unwanted cell type has a characteristically different pH from the extracellular microenvironment, such as particularly the glycocalyx. Because pH cleavage can occur in all cells in the lysozymes, selection of a targeting agent when using a pH-sensitive cleavage site may require, when desired, more specificity. For example, when a pH-sensitive cleavage site is used, a targeting agent that binds only or highly preferably to cancer cells may be desired (such as, for example, an antibody binding to mesothelin for treatment of lung cancer).
In certain embodiments, at least one cleavage site may be cleaved by a complement-dependent cleavage reaction. Once a targeted ATTAC or TEAC binds to the unwanted cell, the patient's complement cascade may be triggered. In such a case, the complement cascade may also be used to cleave the inert binding partner from the first immune cell or T-cell engaging domain by using a cleavage site sensitive to a complement protease. For example, C1r and C1s and the C3 convertases (C4B,2a and C3b,Bb) are serine proteases. C3/C5 and C5 are also complement proteases. Mannose-associated binding proteins (MASP), serine proteases also involved in the complement cascade and responsible for cleaving C4 and C2 into C4b2b (a C3 convertase) may also be used. For example, and without limitation, C1s cleaves YLGRSYKV (SEQ ID NO: 213) and MQLGRX (SEQ ID NO: 214). MASP2 is believed to cleave SLGRKIQI (SEQ ID NO: 215). Complement component C2a and complement factor Bb are believed to cleave GLARSNLDE (SEQ ID NO: 216).
In some embodiments, at least one cleavage site may be cleaved by a protease that is colocalized to the unwanted cell by a targeting moiety that is the same or different from the targeting moiety in the ATTAC or TEAC. For example, any protease may be simultaneously directed to the microenvironment of the unwanted cells by conjugating the protease to a targeting agent that delivers the protease to that location. The targeting agent may be any targeting agent described herein. The protease may be affixed to the targeting agent through a peptide or chemical linker and may maintain sufficient enzymatic activity when bound to the targeting agent.
In some embodiments comprising two cleavage sites, the protease cleavage sites are different. In some embodiments comprising two cleavage sites, the protease cleavage sites are the same.
H. Linkers
In addition to the cleavage site, linkers may optionally be used to attach the separate parts of the ATTAC or TEAC together. By linker, we include any chemical moiety that attaches these parts together. In some embodiments, the linkers may be flexible linkers. Linkers include peptides, polymers, nucleotides, nucleic acids, polysaccharides, and lipid organic species (such as polyethylene glycol). In some embodiments, the linker is a peptide linker. Peptide linkers may be from about 2-100, 10-50, or 15-30 amino acids long. In some embodiments, peptide linkers may be at least 10, at least 15, or at least 20 amino acids long and no more than 80, no more than 90, or no more than 100 amino acids long. In some embodiments, the linker is a peptide linker that has a single or repeating GGGGS (SEQ ID NO: 85), GGGS (SEQ ID NO: 86), GS (SEQ ID NO: 87), GSGGS (SEQ ID NO: 88), GGSG (SEQ ID NO: 89), GGSGG (SEQ ID NO: 90), GSGSG (SEQ ID NO: 91), GSGGG (SEQ ID NO: 92), GGGSG (SEQ ID NO: 93), and/or GSSSG (SEQ ID NO: 94) sequence(s).
In some embodiments, the linker is a maleimide (MPA) or SMCC linker.
Linkers are also described in U.S. Pat. No. 10,035,856.
I. Two-Component TEAC or ATTAC Comprising Copies of Domains or Moieties
In some embodiments, one or both components comprise two copies of one or more domains or moieties. In some embodiments, the first component comprises two copies of one or more domains or moieties. In some embodiments, the second component comprises two copies of one or more domains or moieties. In some embodiments, both components comprise two copies of one or more domains or moieties.
In some embodiments, the first component comprises two copies of a first targeting moiety; two copies of a first T-cell engaging domain; and two copies of a first inert binding partner. In some embodiments, the second component comprises two copies of a second targeting moiety; two copies of a second T-cell engaging domain; and two copies of a second inert binding partner. In some embodiments, a protease cleavage site separates both inert binding partners from their respective T-cell engaging domains.
In some embodiments, the two copies of the targeting moiety are the same. In some embodiments, the two copies of the T-cell engaging domain are the same. In some embodiments, the two copies of the inert binding partner are the same. In some embodiments, the two copies of the protease cleavage site separating the inert binding partners from their respective T-cell engaging domains are the same. In some embodiments, the two copies of a protease cleavage site separating the inert binding partners from their respective T-cell engaging domains are different.
In some embodiments, a component comprising two copies of a first targeting moiety; two copies of a first T-cell engaging domain; and two copies of a first inert binding partner is generated in a cell via Fc pairing, as described in Examples 1 and 2.
J. Single-Component of a Two-Component TEAC or ATTAC
In some embodiments, cancer cells are targeted using a kit or composition comprising a component of a TEAC or ATTAC.
In some embodiments, a component of a TEAC or ATTAC comprising a half-life extending moiety can be administered with another component also comprising a half-life extending moiety.
In some embodiments, a component of a TEAC or ATTAC comprising a half-life extending moiety can be administered with another component that does not comprise a half-life extending moiety. In this way, only one component of a two-component TEAC or ATTAC has a half-life extending moiety.
In some embodiments, a component is a TEAC component. In some embodiments, a component for use in a kit or composition for treating cancer in a patient comprises a first targeted immune cell engaging agent comprising a targeting moiety that binds a tumor antigen expressed by the cancer; an immune cell engaging domain capable of immune cell binding activity when binding another immune cell engaging domain, wherein the other immune cell engaging domain is not part of the first component, and wherein the immune cell engaging domain comprises either a VH domain or VL domain, optionally wherein the immune cell engaging domain comprises a T-cell engaging domain; an inert binding partner for the immune cell engaging domain binding to the immune cell engaging domain such that the immune cell engaging domain does not bind to the other immune cell engaging domain unless the inert binding partner is removed, wherein if the immune cell engaging domain comprises a VH domain, the inert binding partner comprises a VL domain and if the immune cell engaging domain comprises a VL domain, the inert binding partner comprises a VH domain; a half-life extending moiety, wherein the half-life extending moiety is attached (directly or indirectly) to the inert binding partner; and a protease cleavage site separating the immune cell engaging domain and the inert binding partner, wherein the protease cleavage site is capable of releasing the inert binding partner and half-life extending moiety from the immune cell engaging domain in the presence of a protease (1) expressed by the cancer; or (2) colocalized to the cancer by a targeting moiety that binds a tumor antigen expressed by the cancer and that is the same or different from the targeting moiety in the agent, wherein cleavage of the protease cleavage site causes loss of the inert binding partner and allows for complementation with the other immune cell engaging domain that is not part of the agent, further wherein if the immune cell engaging domain comprises a VH domain, the other immune cell engaging domain comprises a VL domain and if the immune cell engaging domain comprises a VL domain, the other immune cell engaging domain comprises a VH domain.
In some embodiments, a component is an ATTAC component. In some embodiments, a component for use in a kit or composition for treating cancer in a patient comprises a first targeted immune cell engaging agent comprising an immune cell selection moiety capable of selectively targeting an immune cell; an immune cell engaging domain capable of immune cell binding activity when binding another immune cell engaging domain, wherein the other immune cell engaging domain is not part of the first component, and wherein the immune cell engaging domain comprises either a VH domain or VL domain, optionally wherein the immune cell engaging domain comprises a T-cell engaging domain; an inert binding partner for the immune cell engaging domain binding to the immune cell engaging domain such that the immune cell engaging domain does not bind to the other immune cell engaging domain unless the inert binding partner is removed, wherein if the immune cell engaging domain comprises a VH domain, the inert binding partner comprises a VL domain and if the immune cell engaging domain comprises a VL domain, the inert binding partner comprises a VH domain; a half-life extending moiety, wherein the half-life extending moiety is attached (directly or indirectly) to the inert binding partner; and a protease cleavage site separating the immune cell engaging domain and the inert binding partner, wherein the protease cleavage site is capable of releasing the inert binding partner and half-life extending moiety from the immune cell engaging domain in the presence of a protease (1) expressed by the cancer; or (2) colocalized to the cancer by a targeting moiety that binds a tumor antigen expressed by the cancer and that is the same or different from the targeting moiety in the agent, wherein cleavage of the protease cleavage site causes loss of the inert binding partner and allows for complementation with the other immune cell engaging domain that is not part of the agent, further wherein if the immune cell engaging domain comprises a VH domain, the other immune cell engaging domain comprises a VL domain and if the immune cell engaging domain comprises a VL domain, the other immune cell engaging domain comprises a VH domain.
This application also describes a TEAC or ATTAC that is comprised in a single agent. In other words, in a single-agent TEAC or ATTAC, all domains necessary for activity are included in a single molecule. A single-agent TEAC or ATTAC could allow dosing of a single agent as a treatment.
In some embodiments, one or more linker attaches different domains in a single-agent TEAC or ATTAC.
In some embodiments, a single-agent TEAC or ATTAC may also be designed to separate into two components after administration to the patient, where the two components are to be released before function. This separation may occur, for example, by protease cleavage in the blood or in the tumor microenvironment. After cleavage, this single-agent now becomes in the patient a two-component TEAC or ATTAC. At this initial point in separation, the inert binding partner is still attached to the T-cell or immune cell engaging domain.
In some embodiments, a single-agent TEAC or ATTAC may be comprised in a single polypeptide chain (i.e., a linker) designed to allow cleavage to generate two separate components, wherein the inert binding partners are on separate components after cleavage. In some embodiments, a single-agent TEAC or ATTAC comprises one or more cleavage site to allow release of one or more inert binding partners, but the other domains of the TEAC or ATTAC are not designed to promote cleavage and separation of other domains besides release of one or more inert binding partners from the T-cell or immune cell engaging domains.
In some embodiments, a cleavage site comprised within a linker covalently binding a first component and the second component is a protease cleavage site. SEQ ID NOs: 1-84 list some exemplary protease cleavage sites that may be used, but the invention is not limited to this set of proteases cleavage sites and other protease cleavage sites may be employed.
In some embodiments, a cleavage site comprised within a linker covalently binding a first component and the second component is a tumor-associated protease cleavage site. A tumor associated protease is one that is associated with a tumor. In some embodiments, a tumor-associated protease has higher expression in the tumor versus other regions of the body. Any protease with expression in a tumor may be used to select a tumor-associated protease cleavage site for the invention.
In some embodiments, a cleavage site comprised within a linker covalently binding a first component and the second component is a cleavage site for a protease found in the blood. Exemplary proteases found in the blood include thrombin, neutrophil elastase, and furin.
A. Single-Agent TEAC
In some embodiments, a TEAC is comprised in a single agent, wherein the agent is not meant to be cleaved outside of the site of action. In this embodiment, there is no protease cleavage site available to separate the agent into separate components of a two-component system, except for the protease cleavage site between the inert binding partner and the T-cell engaging domain. In some embodiments, an agent for treating cancer in a patient comprises a first targeting moiety that binds a tumor antigen expressed by the cancer; a first T-cell engaging domain capable of T-cell binding activity when binding a second T-cell engaging domain, wherein the first T-cell engaging domain comprises either a VH domain or VL domain; a second T-cell engaging domain capable of T-cell binding activity when binding a first T-cell engaging domain, wherein the second T-cell engaging domain comprises either a VH domain or VL domain; a first inert binding partner for the first T-cell engaging domain binding to the first T-cell engaging domain such that the first T-cell engaging domain does not bind to the second T-cell engaging domain unless the inert binding partner is removed, wherein if the first T-cell engaging domain comprises a VH domain, the inert binding partner comprises a VL domain and if the first T-cell engaging domain comprises a VL domain, the inert binding partner comprises a VH domain; and a protease cleavage site separating the first T-cell engaging domain and the first inert binding partner, wherein the protease cleavage site is capable of releasing the inert binding partner from the T-cell engaging domain in the presence of a protease (1) expressed by the cancer or in the cancer microenvironment; or (2) colocalized to the cancer by a targeting moiety that binds a tumor antigen expressed by the cancer and that is the same or different from the targeting moiety in the agent; wherein the first and second T-cell engaging domains are capable of binding a T cell when neither is bound to an inert binding partner, and further wherein if the first T-cell engaging domain comprises a VH domain, the second T-cell engaging domain comprises a VL domain and if the first T-cell engaging domain comprises a VL domain, the second T-cell engaging domain comprises a VH domain.
A single-agent TEAC that is not meant to be cleaved outside of the site of action and that comprises a linker comprising an Fc domain may be referred to as a “IgG-Duo TEAC” or “Duo IgG-TEAC.” Using the term Duo explains that the embodiment employs a single-agent that has all of the targeting and T-cell engaging domains necessary for a functional construct after protease cleavage, such as the construct in
In some embodiments, a single-agent TEAC comprises only one targeting moiety that is capable of targeting the cancer. In some embodiments, a single-agent TEAC further comprises a second targeting moiety that is capable of targeting the cancer.
In some embodiments, a single-agent TEAC (Duo TEAC) is generated via pairing of two TEACs comprising complementary T-cell engaging domains, that have different purification tags, as described in Examples 1 and 2. In some embodiments, TEACs comprising complementary T cell engaging domains and with different purification tags (such as EPEA and histidine) are used to generate a single-agent TEAC. In some embodiments, the two TEACs comprising complementary T cell engaging domains are synthesized on one plasmid separated by an entity such as T2A self cleaving peptide or by using different promoters for each TEAC so that the TEACs are made in the same cells, such as HEK 293T cells. The TEACs would then form the Duo TEAC in the cell by pairing of CH domains to form an Fc domain. In some embodiments, both protein chains contain different purification tags (such as EPEA and histidine) to allow specific purification of the Duo TEAC comprising two different TEACs.
B. Single-Agent ATTAC
In some embodiments, an ATTAC is comprised in a single agent, wherein the agent is not meant to be cleaved outside of the site of action. In this embodiment, there is no protease cleavage site available to separate the agent into separate components of a two-component system, except for the protease cleavage site between the inert binding partner and the immune cell engaging domain. An agent for treating cancer in a patient comprises an immune cell selection moiety capable of selectively targeting an immune cell; a first immune cell engaging domain capable of immune cell binding activity when binding a second immune cell engaging domain, wherein the first immune cell engaging domain comprises either a VH domain or VL domain, optionally wherein the first immune cell engaging domain comprises a T-cell engaging domain; a second immune cell engaging domain capable of immune cell binding activity when binding a first immune cell engaging domain, wherein the second immune cell engaging domain comprises either a VH domain or VL domain, optionally wherein the second immune cell engaging domain comprises a T-cell engaging domain; a first inert binding partner for the first immune cell engaging domain binding to the first immune cell engaging domain such that the first immune cell engaging domain does not bind to the second immune cell engaging domain unless the inert binding partner is removed, wherein if the first immune cell engaging domain comprises a VH domain, the inert binding partner comprises a VL domain and if the first immune cell engaging domain comprises a VL domain, the inert binding partner comprises a VH domain; and a protease cleavage site separating the first immune cell engaging domain and the first inert binding partner, wherein the protease cleavage site is capable of releasing the inert binding partner from the immune cell engaging domain in the presence of a protease (1) expressed by the cancer or in the cancer microenvironment; or (2) colocalized to the cancer by a targeting moiety that binds a tumor antigen expressed by the cancer and that is the same or different from the targeting moiety in the agent; wherein the first and second immune cell engaging domains are capable of binding an immune cell when neither is bound to an inert binding partner, and further wherein if the first immune cell engaging domain comprises a VH domain, the second immune cell engaging domain comprises a VL domain and if the first immune cell engaging domain comprises a VL domain, the second immune cell engaging domain comprises a VH domain.
A single-agent ATTAC that is not meant to be cleaved outside of the site of action and that comprises a linker comprising an Fc domain may be referred to as a “IgG-Duo ATTAC” or “Duo IgG-ATTAC.” Using the term Duo explains that the embodiment employs a single-agent that has all of the targeting and immune cell engaging and binding domains necessary for a functional construct after protease cleavage.
In some embodiments, a single-agent ATTAC comprises only one targeting moiety that is capable of targeting the cancer. In some embodiments, a single-agent ATTAC further comprises a second targeting moiety that is capable of targeting the cancer.
In some embodiments, a single-agent ATTAC is generated and purified in a similar manner as that described for the single-agent TEAC.
C. Single-Agent TEAC or ATTAC Comprising a Second Inert Binding Partner
In some embodiments, a single-agent TEAC or ATTAC further comprises a second inert binding partner for the second immune cell engaging domain binding to the second immune cell engaging domain such that the second immune cell engaging domain does not bind to the first immune cell engaging domain unless the inert binding partner is removed, wherein if the second immune cell or engaging domain comprises a VH domain, the inert binding partner comprises a VL domain and if the second immune cell engaging domain comprises a VL domain, the inert binding partner comprises a VH domain, optionally wherein the second immune cell engaging domain comprises a T-cell engaging domain; and a protease cleavage site separating the second immune cell engaging domain and the second inert binding partner, wherein the protease cleavage site is capable of releasing the inert binding partner from the immune cell engaging domain in the presence of a protease (1) expressed by the cancer or in the cancer microenvironment; or (2) colocalized to the cancer by a targeting moiety that binds a tumor antigen expressed by the cancer and that is the same or different from the targeting moiety in the agent, wherein the first and second immune cell engaging domains are capable of binding an immune cell when neither is bound to an inert binding partner, and further wherein if the first immune cell engaging domain comprises a VH domain, the second immune cell engaging domain comprises a VL domain and if the first immune cell engaging domain comprises a VL domain, the second immune cell engaging domain comprises a VH domain.
D. Single-Agent TEAC or ATTAC Comprising a Half-Life Extending Moiety
In some embodiments, different moieties of a single-agent TEAC or ATTAC are joined via a linker. In some embodiments, a linker attaches the first and second inert binding partners of a single-agent TEAC or ATTAC. In some embodiments, a linker is capable of dissociation with the first and/or second inert binding partner upon cleavage of a protease cleavage sites.
In some embodiments, a linker comprises a half-life extending moiety as described above.
The TEAC or ATTAC may be employed as pharmaceutical compositions. As such, they may be prepared along with a pharmaceutically acceptable carrier. If parenteral administration is desired, for instance, the TEAC or ATTAC may be provided in sterile, pyrogen-free water for injection or sterile, pyrogen-free saline. Alternatively, the TEAC or ATTAC may be provided in lyophilized form for resuspension with the addition of a sterile liquid carrier.
The targeted TEAC and ATTAC as described herein can be made using genetic engineering techniques. Specifically, a nucleic acid may be expressed in a suitable host to produce an ATTAC or TEAC. For example, a vector may be prepared comprising a nucleic acid sequence that encodes the targeted ATTAC or TEAC including all of its component parts and linkers and that vector may be used to transform an appropriate host cell. Other aspects of methods of making and pharmaceutical compositions are described in U.S. Pat. No. 10,035,856. Similar methods can be used to make any of the described embodiments, whether they are two-component or single-component agents.
In some embodiments, one or more nucleic acid molecules encodes the agent or component.
Various regulatory elements may be used in the vector as well, depending on the nature of the host and the manner of introduction of the nucleic acid into the host, and whether episomal maintenance or integration is desired.
Chemical linkage techniques, such as using maleimide or SMCC linkers, may also be employed.
In instances where the binding partner comprises an aptamer, a person of ordinary skill in the art would appreciate how to conjugate an aptamer to a protein, namely the immune cell engaging domain. Aptamers may be conjugated using a thiol linkage or other standard conjugation chemistries. A maleimide, succinimide, or SH group may be affixed to the aptamer to attach it to the immune cell engaging domain.
These agents or components may be used to treat cancer. In some embodiments, this cancer expresses an antigen that one or more targeting moieties can bind.
In some embodiments, a method of treating cancer expressing a tumor antigen that binds the first targeting moiety in a patient comprises administering an agent or component to the patient.
In some embodiments, a method of targeting an immune response of a patient to cancer comprising administering the agent or component to the patient.
In some embodiments, the T cells of the patient express CD3 or TCR and the T cell engaging domain binds CD3 or TCR.
In some embodiments, if the patient has regulatory T cells in the tumor, the selective immune cell engaging agent does not target markers present on regulatory immune cells (including, but not limited to CD4 and CD25).
In some embodiments, a method of treating cancer expressing a tumor antigen in a patient comprises administering a composition comprising a component, wherein the first targeting moiety comprised in the component binds the tumor antigen, and a second component comprising a half-life extending moiety.
In some embodiments, a method of treating cancer expressing a tumor antigen in a patient comprises administering a composition comprising a component, wherein the first targeting moiety comprised in the component binds the tumor antigen, and a second component not comprising a half-life extending moiety.
Thus, components described herein may be provided as kits or compositions that comprise two separate components. In some embodiments, both components of a kit or composition comprise a half-life extending moiety. In some embodiments, one component of a kit or composition comprises a half-life extending moiety, while the other component does not.
The TEAC or ATTAC described herein may be used in a method of treating a disease in a patient characterized by the presence of cancer cells comprising administering a TEAC or ATTAC. Additionally, the agents described herein may also be used in a method of targeting a patient's own immune response to cancer cells comprising administering a TEAC or ATTAC to the patient.
In some embodiments, the patient has cancer or a recognized pre-malignant state. In some embodiments, the patient has undetectable cancer, but is at high risk of developing cancer, including having a mutation associated with an increased risk of cancer. In some embodiments, the patient at high risk of developing cancer has a premalignant tumor with a high risk of transformation. In some embodiments, the patient at high risk of developing cancer has a genetic profile associated with high risk. In some embodiments, the presence of cancer or a pre-malignant state in a patient is determined based on the presence of circulating tumor DNA (ctDNA) or circulating tumor cells. In some embodiments, treatment is pre-emptive or prophylactic. In some embodiments, treatment slow or blocks the occurrence or reoccurrence of cancer.
The amount of the agent administered to the patient may be chosen by the patient's physician so as to provide an effective amount to treat the condition in question. In a two-component TEAC or ATTAC, the first component and the second component of the TEAC or ATTAC may be administered in the same formulation or two different formulations within a sufficiently close period of time to be active in the patient.
The patient receiving treatment may be a human. The patient may be a primate or any mammal. Alternatively, the patient may be an animal, such as a domesticated animal (for example, a dog or cat), a laboratory animal (for example, a laboratory rodent, such as a mouse, rat, or rabbit), or an animal important in agriculture (such as horses, cattle, sheep, or goats).
The cancer may be a solid or non-solid malignancy. In some embodiments, the cancer is a cancer other than a leukemia or lymphoma. In some embodiments, the cancer may be any cancer such as breast cancer, ovarian cancer, endometrial cancer, cervical cancer, bladder cancer, renal cancer, melanoma, lung cancer, prostate cancer, testicular cancer, thyroid cancer, brain cancer, esophageal cancer, gastric cancer, pancreatic cancer, colorectal cancer, liver cancer, leukemia, myeloma, nonHodgkin lymphoma, Hodgkin lymphoma, acute myeloid leukemia, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, lymphoproliferative disorder, myelodysplastic disorder, myeloproliferative disease, and premalignant disease.
In some embodiments, a patient treated with an ATTAC has a tumor characterized by the presence of high levels of regulatory T cells (see Fridman W H et al., Nature Reviews Cancer 12:298-306 (2012) at Table 1). In patients with tumors characterized by a high presence of regulatory T cells, ATTAC therapy may be advantageous over other therapies that non-selectively target T cells, such as unselective BiTEs. In some embodiments, ATTAC therapy avoids engagement of regulatory T cells. In some embodiments, at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of activated T cells are not regulatory T cells. In some embodiments, no regulatory T cells are activated by ATTAC therapy.
In some embodiments, the presence of a biomarker is used to select patients for receiving the TEAC or ATTAC. A wide variety of tumor markers are known in the art, such as those described at www.cancer.gov/about-cancer/diagnosis-staging/diagnosis/tumor-markers-fact-sheet. In some embodiments, the tumor marker is ALK gene rearrangement or overexpression; alpha-fetoprotein; beta-2-microglobulin; beta-human chorionic gonadotropin; BRCA1 or BRCA2 gene mutations; BCR-ABL fusion genes (Philadelphia chromosome); BRAF V600 mutations; C-kit/CD117; CA15-3/CA27.29; CA19-9; CA-125; calcitonin; carcinoembryonic antigen (CEA); CD20; chromogranin A (CgA); chromosomes 3, 7, 17, or 9p21; circulating tumor cells of epithelial origin (CELLSEARCH®); cytokeratin fragment 21-1; EGFR gene mutation analysis; estrogen receptor (ER)/progesterone receptor (PR); fibrin/fibrinogen; HE4; HER2/neu gene amplification or protein overexpression; immunoglobulins; KRAS gene mutation analysis; lactate dehydrogenase; neuron-specific enolase (NSE); nuclear matrix protein 22; programmed death ligand 1 (PD-L1); prostate-specific antigen (PSA); thyroglobulin; urokinase plasminogen activator (uPA); plasminogen activator inhibitor (PAI-1); 5-protein signature (OVA1®); 21-gene signature (Oncotype DX®); or 70-gene signature (Mammaprint®).
The TEAC or ATTAC may be administered alone or in conjunction with other forms of therapy, including surgery, radiation, traditional chemotherapy, or immunotherapy.
In some embodiments, the immunotherapy is checkpoint blockade. Checkpoint blockade refers to agents that inhibit or block inhibitory checkpoint molecules that suppress immune functions. In some embodiments, the checkpoint blockade targets CTLA4, PD1, PD-L1, LAG3, CD40, TIGIT, TIM3, VISTA or HLA-G.
In some embodiments, the immunotherapy is immune cytokines or cytokine fusions. Cytokines refer to cell-signaling proteins naturally made by the body to activate and regulate the immune system. Cytokine fusions refer to engineered molecules comprising all or part of a cytokine. For example, a cytokine fusion may comprise all or part of a cytokine attached to an antibody that allows targeting to a tumor such as Darleukin (see Zegers et al. (2015) Clin. Cancer Res., 21, 1151-60), Teleukin (see WO2018087172).
In some embodiments, the immunotherapy is cancer treatment vaccination. In some embodiments, cancer treatment vaccination boosts the body's natural defenses to fight cancer. These can either be against shared tumor antigens (such as E6, E7, NY-ESO, MUC1, or HER2) or against personalized mutational neoantigens.
The following numbered items provide embodiments as described herein, though the embodiments recited here are not limiting.
Item 1. An agent for treating cancer in a patient comprising: a first component comprising a targeted T-cell engaging agent comprising: a first targeting moiety that binds a tumor antigen expressed by the cancer; a first T-cell engaging domain capable of T-cell engaging activity when binding a second T-cell engaging domain, wherein the second T-cell engaging domain is not part of the first component, and wherein the first T-cell engaging domain comprises either a VH domain or VL domain; a first inert binding partner for the first T-cell engaging domain binding to the first T-cell engaging domain such that the first T-cell engaging domain does not bind to the second T-cell engaging domain unless the inert binding partner is removed, wherein if the first T-cell engaging domain comprises a VH domain, the inert binding partner comprises a VL domain and if the first T-cell engaging domain comprises a VL domain, the inert binding partner comprises a VH domain; a first half-life extending moiety, wherein the first half-life extending moiety is attached (directly or indirectly) to the first inert binding partner; and a protease cleavage site separating the first T-cell engaging domain and the first inert binding partner, wherein the protease cleavage site is capable of releasing the inert binding partner and half-life extending moiety from the T-cell engaging domain in the presence of a protease: (1) expressed by the cancer or in the cancer microenvironment; (2) colocalized to the cancer by a targeting moiety that binds a tumor antigen expressed by the cancer and that is the same or different from the targeting moiety in the agent, and a second component comprising a targeted T-cell engaging agent comprising: a second targeting moiety that binds a tumor antigen expressed by the cancer; a second T-cell engaging domain capable of T-cell binding activity when binding a first T-cell engaging domain, wherein the first T-cell engaging domain is not part of the second component, and wherein the second T-cell engaging domain comprises either a VH domain or VL domain; a second inert binding partner for the second T-cell engaging domain binding to the second T-cell engaging domain such that the second T-cell engaging domain does not bind to the first T-cell engaging domain unless the inert binding partner is removed, wherein if the second T-cell engaging domain comprises a VH domain, the inert binding partner comprises a VL domain and if the second T-cell engaging domain comprises a VL domain, the inert binding partner comprises a VH domain; and a second half-life extending moiety, wherein the second half-life extending moiety is attached (directly or indirectly) to the second inert binding partner; and a protease cleavage site separating the second T-cell engaging domain and the second inert binding partner, wherein the protease cleavage site is capable of releasing the inert binding partner and half-life extending moiety from the T-cell engaging domain in the presence of a protease: (1) expressed by the cancer or in the cancer microenvironment; (2) colocalized to the cancer by a targeting moiety that binds a tumor antigen expressed by the cancer and that is the same or different from the targeting moiety in the agent, wherein the first and second T-cell engaging domains are capable of binding a T cell when neither is bound to an inert binding partner, and further wherein if the first T-cell engaging domain comprises a VH domain, the second T-cell engaging domain comprises a VL domain and if the first T-cell engaging domain comprises a VL domain, the second T-cell engaging domain comprises a VH domain.
Item 2. An agent for treating cancer in a patient comprising: a first component comprising a targeted immune cell engaging agent comprising: a targeting moiety capable of targeting the cancer; a first immune cell engaging domain capable of immune engaging activity when binding a second immune cell engaging domain, wherein the second immune cell engaging domain is not part of the first component, optionally wherein the first immune cell engaging domain comprises a T-cell engaging domain; a first inert binding partner for the first immune cell engaging domain binding to the first immune cell engaging domain such that the first immune cell engaging domain does not bind to the second immune cell engaging domain unless the inert binding partner is removed, wherein if the first immune cell engaging domain comprises a VH domain, the inert binding partner comprises a VL domain and if the first immune cell engaging domain comprises a VL domain, the inert binding partner comprises a VH domain; a first half-life extending moiety, wherein the first half-life extending moiety is attached (directly or indirectly) to the first inert binding partner; and a protease cleavage site separating the first immune cell engaging domain and the first inert binding partner, wherein the protease cleavage site is capable of releasing the inert binding partner and half-life extending moiety from the immune cell engaging domain in the presence of a protease: (1) expressed by the cancer or in the cancer microenvironment; or (2) colocalized to the cancer by a targeting moiety that binds a tumor antigen expressed by the cancer and that is the same or different from the targeting moiety in the agent, and a second component comprising a selective immune cell engaging agent comprising: an immune cell selection moiety capable of selectively targeting an immune cell; a second immune cell engaging domain capable of immune cell engaging activity when binding the first immune cell engaging domain, wherein the first and second immune cell engaging domains are capable of binding when neither is bound to an inert binding partner, optionally wherein the second immune cell engaging domain comprises a immune cell engaging domain; a second inert binding partner for the second immune cell engaging domain binding to the second immune cell engaging domain such that the second immune cell engaging domain does not bind to the first immune cell engaging domain unless the inert binding partner is removed, wherein if the second immune cell engaging domain comprises a VH domain, the inert binding partner comprises a VL domain and if the second immune cell engaging domain comprises a VL domain, the inert binding partner comprises a VH domain; and a second half-life extending moiety, wherein the second half-life extending moiety is attached (directly or indirectly) to the second inert binding partner; a protease cleavage site separating the second immune cell engaging domain and the second inert binding partner, wherein the protease cleavage site is capable of releasing the inert binding partner and half-life extending moiety from the immune cell engaging domain in the presence of a protease: (1) expressed by the cancer or in the cancer microenvironment; or (2) colocalized to the cancer by a targeting moiety that binds a tumor antigen expressed by the cancer and that is the same or different from the targeting moiety in the agent, wherein the first and second immune cell engaging domains are capable of binding an immune cell when neither is bound to an inert binding partner, and further wherein if the first immune cell engaging domain comprises a VH domain, the second immune cell engaging domain comprises a VL domain and if the first immune cell engaging domain comprises a VL domain, the second immune cell engaging domain comprises a VH domain.
Item 3. A component for use in a kit or composition for treating cancer in a patient comprising a first targeted immune cell engaging agent comprising: a targeting moiety that binds a tumor antigen expressed by the cancer; an immune cell engaging domain capable of immune cell binding activity when binding another immune cell engaging domain, wherein the other immune cell engaging domain is not part of the first component, and wherein the immune cell engaging domain comprises either a VH domain or VL domain, optionally wherein the immune cell engaging domain comprises a T-cell engaging domain; an inert binding partner for the immune cell engaging domain binding to the immune cell engaging domain such that the immune cell engaging domain does not bind to the other immune cell engaging domain unless the inert binding partner is removed, wherein if the immune cell engaging domain comprises a VH domain, the inert binding partner comprises a VL domain and if the immune cell engaging domain comprises a VL domain, the inert binding partner comprises a VH domain; a half-life extending moiety, wherein the half-life extending moiety is attached (directly or indirectly) to the inert binding partner; and a protease cleavage site separating the immune cell engaging domain and the inert binding partner, wherein the protease cleavage site is capable of releasing the inert binding partner and half-life extending moiety from the immune cell engaging domain in the presence of a protease: (1) expressed by the cancer; or (2) colocalized to the cancer by a targeting moiety that binds a tumor antigen expressed by the cancer and that is the same or different from the targeting moiety in the agent, wherein cleavage of the protease cleavage site causes loss of the inert binding partner and allows for complementation with the other immune cell engaging domain that is not part of the agent, further wherein if the immune cell engaging domain comprises a VH domain, the other immune cell engaging domain comprises a VL domain and if the immune cell engaging domain comprises a VL domain, the other immune cell engaging domain comprises a VH domain.
Item 4. A component for use in a kit or composition for treating cancer in a patient comprising a first targeted immune cell engaging agent comprising: an immune cell selection moiety capable of selectively targeting an immune cell; an immune cell engaging domain capable of immune cell binding activity when binding another immune cell engaging domain, wherein the other immune cell engaging domain is not part of the first component, and wherein the immune cell engaging domain comprises either a VH domain or VL domain, optionally wherein the immune cell engaging domain comprises a T-cell engaging domain; an inert binding partner for the immune cell engaging domain binding to the immune cell engaging domain such that the immune cell engaging domain does not bind to the other immune cell engaging domain unless the inert binding partner is removed, wherein if the immune cell engaging domain comprises a VH domain, the inert binding partner comprises a VL domain and if the immune cell engaging domain comprises a VL domain, the inert binding partner comprises a VH domain; a half-life extending moiety, wherein the half-life extending moiety is attached (directly or indirectly) to the inert binding partner; and a protease cleavage site separating the immune cell engaging domain and the inert binding partner, wherein the protease cleavage site is capable of releasing the inert binding partner and half-life extending moiety from the immune cell engaging domain in the presence of a protease: (1) expressed by the cancer; or (2) colocalized to the cancer by a targeting moiety that binds a tumor antigen expressed by the cancer and that is the same or different from the targeting moiety in the agent, wherein cleavage of the protease cleavage site causes loss of the inert binding partner and allows for complementation with the other immune cell engaging domain that is not part of the agent, further wherein if the immune cell engaging domain comprises a VH domain, the other immune cell engaging domain comprises a VL domain and if the immune cell engaging domain comprises a VL domain, the other immune cell engaging domain comprises a VH domain.
Item 5. An agent for treating cancer in a patient comprising: a first targeting moiety that binds a tumor antigen expressed by the cancer; a first T-cell engaging domain capable of T-cell binding activity when binding a second T-cell engaging domain, wherein the first T-cell engaging domain comprises either a VH domain or VL domain; a second T-cell engaging domain capable of T-cell binding activity when binding a first T-cell engaging domain, wherein the second T-cell engaging domain comprises either a VH domain or VL domain; a first inert binding partner for the first T-cell engaging domain binding to the first T-cell engaging domain such that the first T-cell engaging domain does not bind to the second T-cell engaging domain unless the inert binding partner is removed, wherein if the first T-cell engaging domain comprises a VH domain, the inert binding partner comprises a VL domain and if the first T-cell engaging domain comprises a VL domain, the inert binding partner comprises a VH domain; and a protease cleavage site separating the first T-cell engaging domain and the first inert binding partner, wherein the protease cleavage site is capable of releasing the inert binding partner from the T-cell engaging domain in the presence of a protease: (1) expressed by the cancer or in the cancer microenvironment; or (2) colocalized to the cancer by a targeting moiety that binds a tumor antigen expressed by the cancer and that is the same or different from the targeting moiety in the agent; wherein the first and second T-cell engaging domains are capable of binding a T cell when neither is bound to an inert binding partner, and further wherein if the first T-cell engaging domain comprises a VH domain, the second T-cell engaging domain comprises a VL domain and if the first T-cell engaging domain comprises a VL domain, the second T-cell engaging domain comprises a VH domain.
Item 6. An agent for treating cancer in a patient comprising: an immune cell selection moiety capable of selectively targeting an immune cell; a first immune cell engaging domain capable of immune cell binding activity when binding a second immune cell engaging domain, wherein the first immune cell engaging domain comprises either a VH domain or VL domain, optionally wherein the first immune cell engaging domain comprises a T-cell engaging domain; a second immune cell engaging domain capable of immune cell binding activity when binding a first immune cell engaging domain, wherein the second immune cell engaging domain comprises either a VH domain or VL domain, optionally wherein the second immune cell engaging domain comprises a T-cell engaging domain; a first inert binding partner for the first immune cell engaging domain binding to the first immune cell engaging domain such that the first immune cell engaging domain does not bind to the second immune cell engaging domain unless the inert binding partner is removed, wherein if the first immune cell engaging domain comprises a VH domain, the inert binding partner comprises a VL domain and if the first immune cell engaging domain comprises a VL domain, the inert binding partner comprises a VH domain; and a protease cleavage site separating the first immune cell engaging domain and the first inert binding partner, wherein the protease cleavage site is capable of releasing the inert binding partner from the immune cell engaging domain in the presence of a protease: (1) expressed by the cancer or in the cancer microenvironment; or (2) colocalized to the cancer by a targeting moiety that binds a tumor antigen expressed by the cancer and that is the same or different from the targeting moiety in the agent; wherein the first and second immune cell engaging domains are capable of binding an immune cell when neither is bound to an inert binding partner, and further wherein if the first immune cell engaging domain comprises a VH domain, the second immune cell engaging domain comprises a VL domain and if the first immune cell engaging domain comprises a VL domain, the second immune cell engaging domain comprises a VH domain.
Item 7. The agent of any one of items 5 or 6, wherein the agent further comprises a second targeting moiety that is capable of targeting the cancer.
Item 8. The agent of any one of items 5-7 further comprising: a second inert binding partner for the second immune cell engaging domain binding to the second immune cell engaging domain such that the second immune cell engaging domain does not bind to the first immune cell engaging domain unless the inert binding partner is removed, wherein if the second immune cell or engaging domain comprises a VH domain, the inert binding partner comprises a VL domain and if the second immune cell engaging domain comprises a VL domain, the inert binding partner comprises a VH domain, optionally wherein the second immune cell engaging domain comprises a T-cell engaging domain; and a protease cleavage site separating the second immune cell engaging domain and the second inert binding partner, wherein the protease cleavage site is capable of releasing the inert binding partner from the immune cell engaging domain in the presence of a protease: (1) expressed by the cancer or in the cancer microenvironment; or (2) colocalized to the cancer by a targeting moiety that binds a tumor antigen expressed by the cancer and that is the same or different from the targeting moiety in the agent, wherein the first and second immune cell engaging domains are capable of binding an immune cell when neither is bound to an inert binding partner, and further wherein if the first immune cell engaging domain comprises a VH domain, the second immune cell engaging domain comprises a VL domain and if the first immune cell engaging domain comprises a VL domain, the second immune cell engaging domain comprises a VH domain.
Item 9. The agent of item 8, wherein a linker attaches the first and second inert binding partners.
Item 10. The agent of item 9, wherein the linker comprises a half-life extending moiety.
Item 11. The agent of any one of items 9-10, wherein the linker is capable of dissociation with the first and/or second inert binding partner upon cleavage of the protease cleavage sites.
Item 12. The agent of any one of items 1-11, wherein the first and/or second half-life extending moiety is directly attached to the first and/or second inert binding partner.
Item 13. The agent of any one of items 1-11, wherein the first and/or second half-life extending moiety is indirectly attached to the first and/or second inert binding partner via a linker.
Item 14. The agent of any one of items 1-2, wherein the first component comprises: two copies of a first targeting moiety; two copies of a first immune or T-cell engaging domain; and two copies of a first inert binding partner, wherein a protease cleavage site separates both inert binding partners from their respective immune or T-cell engaging domains.
Item 15. The agent of item 14, wherein one end of the half-life extending moiety is attached (directly or indirectly) to one copy of the first inert binding partner and the other end of the half-life extending moiety is attached (directly or indirectly) to the other copy of the first inert binding partner.
Item 16. The agent of any one of items 1-2, 14, or 15, wherein the second component comprises: two copies of a second targeting moiety; two copies of a second immune or T-cell engaging domain; and two copies of a second inert binding partner, wherein a protease cleavage sites separates both inert binding partners from their respective immune or T-cell engaging domains.
Item 17. The agent of item 16, wherein one end of the half-life extending moiety is attached (directly or indirectly) to one copy of the second inert binding partner and the other end of the half-life extending moiety is attached (directly or indirectly) to the other copy of the second inert binding partner.
Item 18. The agent of any one of items 14-17, wherein the two copies of the targeting moiety are the same.
Item 19. The agent of any one of items 14-18, wherein the two copies of the immune or T-cell engaging domain are the same.
Item 20. The agent of any one of items 14-19, wherein the two copies of the inert binding partner are the same.
Item 21. The agent of any one of items 14-20, wherein the two copies of the protease cleavage site separating the inert binding partners from their respective immune or T-cell engaging domains are the same.
Item 22. The agent of any one of items 14-20, wherein the two copies of a protease cleavage site separating the inert binding partners from their respective immune or T-cell engaging domains are different.
Item 23. The agent or component of any one of items 1-22, wherein the half-life is decreased after dissociation of one or more half-life extending moieties.
Item 24. The agent of any one of items 1-2 or 12-23, wherein the half-life of the first and/or second component is longer than the half-life of a complex formed by the association of the first and second immune cell or T-cell engaging domains in the form capable of binding to an immune or T cell.
Item 25. The agent of any one of items 1-2 or 12-24, wherein the first component and/or second component has a half-life greater or equal to 2 days, 4 days, or 7 days.
Item 26. The agent or component of any one of items 3-11, wherein the agent or component has a half-life greater or equal to 2 days, 4 days, or 7 days.
Item 27. The agent of any one of items 1, 2, 8-11 or 14-26, wherein the protease cleavage sites are different.
Item 28. The agent of any one of items 8-11 or 14-26, wherein the protease cleavage sites are the same.
Item 29. The agent or component of any one of items 1-28, wherein one or more protease cleavage sites are cleaved by a protease expressed by the cancer.
Item 30. The agent or component of any one of items 1-29, wherein one or more protease cleavage sites are cleaved by a protease that is colocalized to the cancer by a targeting moiety that binds a tumor antigen expressed by the cancer and that is the same or different from the targeting moiety in the agent.
Item 31. The agent or component of any one of items 1-30, wherein one or more first and second inert binding partners are capable of dissociation once at least one protease cleavage site for each inert binding partner has been cleaved and after dissociation the two immune cell or T-cell engaging domains that had been bound by the inert binding partners are capable of binding to each other and exhibiting immune cell or T-cell binding activity.
Item 32. The agent or component of any one of items 1-31, wherein one or more half-life extending moieties are capable of dissociation together with one or more inert binding partner to which it is attached.
Item 33. The agent or component of any one of items 1-4 or 10-32, wherein the one or more half-life extending moieties comprise all or part of an immunoglobulin constant (Fc) domain, serum albumin, serum albumin binding protein, an unstructured protein, and/or PEG.
Item 34. The agent or component of item 33, wherein the one or more half-life extending moieties comprise all or part of an immunoglobulin Fc domain.
Item 35. The agent or component of item 34, wherein the Fc domain comprises the sequence of a human immunoglobulin.
Item 36. The agent or component of item 34, wherein the immunoglobulin is IgG.
Item 37. The agent or component of item 36, wherein the IgG is IgG1, IgG2, or IgG4.
Item 38. The agent or component of item 34, wherein the Fc domain comprises a naturally occurring sequence.
Item 39. The agent or component of item 34, wherein the Fc domain comprises one or more mutations as compared to a naturally occurring sequence.
Item 40. The agent or component of item 39, wherein the Fc domain is an Fc domain with a longer half-life compared to a naturally occurring sequence.
Item 41. The agent or component of item 40, wherein the Fc domain with a longer half-life has increased FcRn binding.
Item 42. The agent or component of item 41, wherein the increased FcRn binding is measured at pH 6.0.
Item 43. The agent or component of item 40, wherein the Fc domain with a longer half-life comprises M252Y/S254T/T256E substitutions.
Item 44. The agent or component of item 40, wherein the Fc domain with a longer half-life comprises M428L/N434S substitutions.
Item 45. The agent or component of item 33, wherein one or more half-life extending moiety comprise all or part of serum albumin.
Item 46. The agent or component of item 45, wherein the serum albumin is human.
Item 47. The agent or component of item 33, wherein one or more half-life extending moiety comprise all or part of a serum albumin binding protein.
Item 48. The agent or component of item 47, wherein the serum albumin binding protein is a DARPin, a nanobody, a single-chain variable fragment (scFv), or an antigen-binding fragment (Fab).
Item 49. The agent or component of item 33, wherein the serum albumin binding protein comprises all or part of an albumin binding domain.
Item 50. The agent or component of item 33, wherein one or more half-life extending moiety comprise all or part of an unstructured protein.
Item 51. The agent or component of item 50, wherein the unstructured protein is an unstructured hydrophilic, biodegradable protein polymer.
Item 52. The agent or component of item 51, wherein the unstructured protein is XTEN.
Item 53. The agent or component of item 33, wherein one or more half-life extending moiety comprise all or part of PEG.
Item 54. The agent of any one of items 1-2 and 12-53, wherein the first and second half-life extending moieties are different.
Item 55. The agent of any one of items 1-2 and 12-53, wherein the first and second half-life extending moieties are the same.
Item 56. The agent of any one of items 1-2 and 12-55, wherein the first component is not covalently bound to the second component.
Item 57. The agent of any one of items 1-2 and 12-55, wherein the first component is covalently bound to the second component.
Item 58. The agent of item 57, wherein the first component is covalently bound to the second component by a linker comprising a protease cleavage site.
Item 59. The agent or component of item 2, 4, or 6-58, wherein the immune cell selection moiety capable of selectively targeting an immune cell selectively targets a T cell, a macrophage, a natural killer cell, a neutrophil, an eosinophil, a basophil, a γδ T cell, a natural killer T cell (NKT cells), or an engineered immune cell.
Item 60. The agent or component of item 59, wherein the immune cell selection moiety capable of selectively targeting an immune cell selectively targets a T cell, optionally where the T cell is a CD8+ or CD4+ T cell.
Item 61. The agent or component of item 59, wherein the immune cell selection moiety targets CD8, CD4, or CXCR3, or does not specifically bind regulatory T cells.
Item 62. The agent or component of any one of items 59-61, wherein the immune cell selection moiety comprises an aptamer or an antibody or antigen-specific binding fragment thereof.
Item 63. The agent or component of item 62, wherein the aptamer or antibody or antigen-specific binding fragment thereof specifically binds an antigen on a T cell.
Item 64. The agent or component of any one of items 1-2 or 5-63, wherein the first and second T-cell or immune cell engaging domains are capable of binding CD3 or the T cell receptor (TCR) when neither is bound to an inert binding partner.
Item 65. The agent or component of any one of items 1-2 or 5-64, wherein the first and second T-cell or immune cell engaging domains are capable of forming a Fv when not bound to an inert binding partner.
Item 66. The agent or component of any one of items 1-5 or 7-65, wherein one or more targeting moieties are an antibody or antigen-binding fragment thereof.
Item 67. The agent or component of item 66, wherein the antibody or antigen-binding fragment thereof is (i) specific for any of 4-1BB, 5T4, ACVRL1, ALK1, AXL, B7-H3, BCMA, c-MET, CD133, C4.4a, CA6, CA9, Cadherin-6, CD123, CD133, CD138, CD19, CD20, CD22, CD25, CD27L, CD30, CD33, CD37, CD38, CD44v6, CD56, CD70, CD74(TROP2), CD79b, CEA, CEACAM5, cKit, CLL-1, Cripto, CS1, DLL3, EDNRB, EFNA4, EGFR, EGFRvIII, ENPP3, EpCAM, EPHA2, FGFR2, FGFR3, FLT3, FOLR, FOLR1, GD2, gpA33, GPC3, GPNMB, GUCY2C, HER2, HER3, HLAA2, IGF1-r, IL13RA2, Integrin alpha, LAMP-1, LewisY, LIV-1, LRRC15, MMP9, MSLN, MUC1, MUC16, NaPi2b, Nectin-4, NOTCH3, p-CAD, PD-L1, PSMA, PTK7, ROR1, SLC44A4, SLITRK6, SSTR2, STEAP1, TAG72, TF, TIM-1, or TROP-2, or (ii) an anti-epidermal growth factor receptor antibody; an anti-Her2 antibody; an anti-CD20 antibody; an anti-CD22 antibody; an anti-CD70 antibody; an anti-CD33 antibody; an anti-MUC1 antibody; an anti-CD40 antibody; an anti-CD74 antibody; an anti-P-cadherin antibody; an anti-EpCAM antibody; an anti-CD138 antibody; an anti-E-cadherin antibody; an anti-CEA antibody; an anti-FGFR3 antibody; an anti-mucin core protein antibody; an anti-transferrin antibody; an anti-gp95/97 antibody; an anti-p-glycoprotein antibody; an anti-TRAIL-R1 antibody; an anti-DR5 antibody; an anti-IL-4 antibody; an anti-IL-6 antibody; an anti-CD19 antibody; an anti-PSMA antibody; an anti-PSCA antibody; an anti-Cripto antibody; an anti-PD-L1 antibody; an anti-IGF-1R antibody; an anti-CD38 antibody; an anti-CD133 antibody; an anti-CD123 antibody; an anti-CDE49d antibody; an anti-glypican 3 antibody; an anti-cMET antibody; or an anti-IL-13R antibody.
Item 68. The agent or component of item 66, wherein the antibody or antigen-binding fragment comprises all or part of the amino acid sequence of 1C1, (GS) 5745, ABBV-085, ABBV-399, ABBV-838, AbGn-107, ABT-414, ADCT-301, ADCT-402, AGS-16C3F, AGS62P1, AGS67E, AMG 172d, AMG 595d, Andecaliximab, Anetumab ravtansine, ARX788, ASG-15MEd, ASG-5MEk, Atezolizumab, AVE1642, AVE9633e, Avelumab, BAY1129980, BAY1187982e, BAY79-4620b, BIIB015d, Bivatuzumab mertansineb, BMS-986148, Brentuximab vedotin, Cantuzumab mertansine, CC49, CDX-014, Cirmtuzumab, Coltuximab ravtansine, DEDN6526Ae, Denintuzumab mafodotin, Depatuxizumab, DFRF4539Ad, DMOT4039Ae, DS-8201A, Durvalumab, Enfortumab vedotin, Farletuzumab, FLYSYN, Gatipotuzumab, Gemtuzumab ozogamicin, Glembatumumab vedotin, GSK2857916, HKT288, Hu3F8, HuMax-AXL-ADC, IDEC-159, IMGN289b, IMGN388a, IMGN529, Indatuximab ravtansine, Inotuzumab ozogamicin, Istiratumab, Labetuzumab govitecan, Lifastuzumab vedotin, LOP628h, Lorvotuzumab mertansine, LY3076226, MCLA-117 (CLEC-12AxCD3), MDX-1203d, MEDI-4276, MEDI-547b, Milatuzumab-doxorubicin, Mirvetuximab soravtansine, MLN0264, MLN2704e, MM-302i, Mosunetuzumab, MOv18 IgE, Ocrelizumab, Oportuzumab, Patritumab, PCA-062, PF-03446962, PF-06263507a, PF-06647020, PF-06647263, PF-06650808d, Pinatuzumab vedotin, Polatuzumab vedotin, PSMA ADC 301c, RC48-ADC, Rituximab, Rovalpituzumab tesirine, Sacituzumab, Sacituzumab govitecan, SAR408701, SAR428926, SAR566658, SC-002, SC-003, SGN-15a, SGN-CD123A, SGN-CD19B, SGN-CD70A, SGN-LIV1A, Sofituzumab vedotin, Solitomab, SSTR2xCD3 XmAb18087, STRO-002, SYD-985, Talacotuzumab, Tisotumab vedotin, Trastuzumab emtansine, U3-1402, Ublituximab, Vadastuximab talirine, Vandortuzumab vedotin, Vorsetuzumab mafodotin, XMT-1522, or Zenocutuzumab.
Item 69. The agent or component of any one of items 1-68, wherein one or more targeting moieties are an aptamer.
Item 70. The agent or component of item 69, wherein the aptamer comprises DNA.
Item 71. The agent or component of item 69, wherein the aptamer comprises RNA.
Item 72. The agent or component of any one of items 69-71, wherein the aptamer is single-stranded.
Item 73. The agent or component of any one of items 69-72, wherein the aptamer is a target cell-specific aptamer chosen from a random candidate library.
Item 74. The agent or component of any one of items 69-73, wherein the aptamer is an anti-EGFR aptamer.
Item 75. The agent or component of any one of items 69-74, wherein the aptamer binds to the antigen on the cancer cell with a Kd from 1 picomolar to 500 nanomolar.
Item 76. The agent or component of item 75, wherein the aptamer binds to the cancer with a Kd from 1 picomolar to 100 nanomolar.
Item 77. The agent or component of any one of items 1-5 or 7-76, wherein one or more targeting moiety comprise IL-2, IL-4, IL-6, α-MSH, transferrin, folic acid, EGF, TGF, PD1, IL-13, stem cell factor, insulin-like growth factor (IGF), or CD40.
Item 78. The agent or component of any one of items 1-5 or 7-77, wherein one or more targeting moiety comprise a full-length sequence of IL-2, IL-4, IL-6, α-MSH, transferrin, folic acid, EGF, TGF, PD1, IL-13, stem cell factor, insulin-like growth factor (IGF), or CD40.
Item 79. The agent or component of any one of items 1-5 or 7-77, wherein one or more targeting moiety comprise a truncated form, analog, variant, or derivative of IL-2, IL-4, IL-6, α-MSH, transferrin, folic acid, EGF, TGF, PD1, IL-13, stem cell factor, insulin-like growth factor (IGF), or CD40.
Item 80. The agent or component of any one of items 1-5 or 7-79, wherein one or more targeting moiety bind a target on the cancer comprising IL-2 receptor, IL-4, IL-6, melanocyte stimulating hormone receptor (MSH receptor), transferrin receptor (TR), folate receptor 1 (FOLR), folate hydroxylase (FOLH1), EGF receptor, PD-L1, PD-L2, IL-13R, CXCR4, IGFR, or CD40L.
Item 81. The agent of any one of items 1, 7-58, or 64-80, wherein the first and second targeting moieties bind the same antigen.
Item 82. The agent of item 81, wherein the first and second targeting moieties bind the same epitope.
Item 83. The agent of item 82, wherein the first and second targeting moieties are the same.
Item 84. The agent of any one of items 1, 7-58, or 64-80, wherein the first and second targeting moieties are different.
Item 85. The agent of item 84, wherein the first and second targeting moieties bind different antigens.
Item 86. The agent of item 84, wherein the first and second targeting moieties bind different epitopes of the same antigen.
Item 87. A method of treating cancer expressing a tumor antigen that binds the first targeting moiety in a patient comprising administering the agent or component of any one of items 1-86 to the patient.
Item 88. The method of item 87, wherein the cancer expressing a tumor antigen that binds the first targeting moiety is any one of breast cancer, ovarian cancer, endometrial cancer, cervical cancer, bladder cancer, renal cancer, melanoma, lung cancer, prostate cancer, testicular cancer, thyroid cancer, brain cancer, esophageal cancer, gastric cancer, pancreatic cancer, colorectal cancer, liver cancer, leukemia, myeloma, nonHodgkin lymphoma, Hodgkin lymphoma, acute myeloid leukemia, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, lymphoproliferative disorder, myelodysplastic disorder, myeloproliferative disease or premalignant disease.
Item 89. A method of targeting an immune response of a patient to cancer comprising administering the agent or component of any one of items 87-88 to the patient.
Item 90. The method of any one of items 87-89, wherein the T cells express CD3 or TCR and the T cell engaging domain binds CD3 or TCR.
Item 91. The method of any one of items 2, 4, or 6-80, wherein if the patient has regulatory T cells in the tumor, the selective immune cell engaging agent does not target markers present on regulatory immune cells (including, but not limited to CD4 and CD25).
Item 92. A method of treating cancer expressing a tumor antigen in a patient comprising administering a composition comprising the component of any one of items 3, 4, 23, 26, 29-53, or 59-80, wherein the first targeting moiety binds the tumor antigen and a second component comprising a half-life extending moiety.
Item 93. A method of treating cancer expressing a tumor antigen in a patient comprising administering a composition comprising the component of any one of items 3, 4, 23, 26, 29-53, or 59-80, wherein the first targeting moiety binds the tumor antigen and a second component not comprising a half-life extending moiety.
Item 94. One or more nucleic acid molecules encoding the agent or component of any of items 1-80.
Item 95. An agent for treating cancer in a patient comprising: a first component comprising: a targeting antibody or antigen-specific binding fragment thereof that binds a tumor antigen expressed by the cancer; a first VH or VL domain capable of T-cell engaging activity when binding a second VH or VL domain, wherein the second VH or VL domain is not part of the first component; a first inert binding partner for the first VH or VL domain such that the first VH or VL domain does not bind to the second VH or VL domain unless the inert binding partner is removed, wherein a first VH domain can bind an inert binding partner comprising a VL domain and a first VL domain can bind an inert binding partner comprising a VH domain; a first half-life extending moiety, wherein the first half-life extending moiety is attached (directly or indirectly) to the first inert binding partner; and a protease cleavage site separating the first VH or VL domain and the first inert binding partner, wherein the protease cleavage site is capable of releasing the inert binding partner and half-life extending moiety from the rest of the agent in the presence of a protease: (1) expressed by the cancer or in the cancer microenvironment; (2) colocalized to the cancer by a targeting antibody or antigen-specific binding fragment thereof that binds a tumor antigen expressed by the cancer and that is the same or different from the targeting antibody or antigen-specific binding fragment thereof in the agent, and a second component comprising: a second targeting antibody or antigen-specific binding fragment thereof that binds a tumor antigen expressed by the cancer; a second VH or VL domain capable of T-cell binding activity when binding a first VH or VL domain, wherein the first VH or VL domain is not part of the second component; a second inert binding partner for the second VH or VL domain binding such that the second VH or VL domain does not bind to the first VH or VL domain unless the inert binding partner is removed, wherein if the second VH or VL domain comprises a VH domain, the inert binding partner comprises a VL domain and if the second VH or VL domain comprises a VL domain, the inert binding partner comprises a VH domain; and a second half-life extending moiety, wherein the second half-life extending moiety is attached (directly or indirectly) to the second inert binding partner; a protease cleavage site separating the second VH or VL domain and the second inert binding partner, wherein the protease cleavage site is capable of releasing the inert binding partner and half-life extending moiety from the rest of the agent in the presence of a protease: (1) expressed by the cancer or in the cancer microenvironment; (2) colocalized to the cancer by a targeting antibody or antigen-specific binding fragment thereof that binds a tumor antigen expressed by the cancer and that is the same or different from the targeting antibody or antigen-specific binding fragment thereof in the agent, wherein the first and second VH or VL domains are capable of binding a T cell when neither is bound to an inert binding partner, and further wherein if the first VH or VL domain comprises a VH domain, the second VH or VL domain comprises a VL domain and if the first VH or VL domain comprises a VL domain, the second VH or VL comprises a VH domain.
Two-component dual IgG TEACs were developed wherein each component of the two-component system comprises an IgG TEAC. The dual IgG TEAC was designed comprising a first component that was an IgG TEAC comprising two targeting moieties that are each an anti-CD33 antibody and a second component that was an IgG TEAC comprising two targeting moieties that are each an anti-CD123 antibody. Each IgG TEAC also comprises two copies of T-cell engaging domain, two copies of an inert binding partners, and two copies of a cleavage site between the T-cell engaging domains and the inert binding partners.
Further, both the first and second components together also comprise a linker comprising a half-life extending moiety. The half-life extending moiety comprises an Fc domain from IgG4, wherein the Fc domain is directly linked to the inert binding partner. When expressed in HEK293T cells, one copy of the TEAC (CD33 binding region (scFv)-anti-CD3 domain-cleavage sequence-inert binding partner-Fc domain) will recombine with a second copy of the TEAC via covalent interactions of the Fc domains to form the IgG TEAC.
As an example, a TEAC was composed of VH-VL specific for a tumor antigen (either CD33 or CD123) linked to a second scFv composed of VL-VH with the VL specific for CD3 and the VH being the inert binding partner or with the VH specific for CD3 and VL being the inert binding partner (SEQ ID NO: 199 for anti-CD33 TEAC component and SEQ ID NO: 200 for anti-CD123 TEAC component). For the IgG TEACs, conventional TEAC components were used as the base and the CH domains from IgG4 were added to allow pairing. This made the resulting IgG TEAC into a more conventional antibody shape with two TEAC acting as the “arms,” and the CH regions acting as the conventional CH regions of an antibody.
When the TEACs were produced in the IgG format in 293T cells, the DNA plasmid that was transfected into the cells contained one single sequence that was produced multiple times, and the proteins paired to form a single antibody-like protein, the IgG TEAC. Each IgG TEAC was generated in this manner separately in a different population of cells, and then the CD33 and CD123 IgG TEACs could be used together as a pair of two different IgG TEACs, i.e., a Dual IgG TEAC.
A single-agent TEAC (Duo TEAC) comprising a half-life extending moiety was also designed that comprises SEQ ID NOs: 199 and 200, such that it comprises one targeting moiety which is an anti-CD33 antibody and one targeting moiety that is an anti-CD123 antibody.
The first and second inert binding partners of the single-agent TEAC were connected via a linker comprising a half-life extending moiety. The half-life extending moiety comprises an Fc domain from IgG4 wherein the inert binding partner is directly linked to the Fc domain.
For the Duo-TEAC, two different sequences were needed that coded for each “arm” or half of the antibody-like protein. Therefore, the construct comprised a CH domain, followed by the inert binding domain, the anti-CD3 domain, and then the tumor binding domain (e.g., CD33). In the same DNA construct, a second sequence was added for a construct that comprised a CH domain followed by the inert binding partner, the complementary anti-CD3 domain, and then the second tumor binding domain (e.g., CD123).
When expressed in HEK293T cells, one chain of the protein (CD33 binding region (scFv)-anti-CD3 domain-cleavage sequence-inert binding partner-Fc domain) will recombine with a second chain of the protein (containing the CD123 binding domain) via covalent interactions of the CH domains to form the Duo IgG TEAC. Once this DNA construct was transfected into the 293T cells, both proteins were produced by the cells and there would be three possible protein pairs (i) CD33 and CD33; (ii) CD123 and CD123; (iii) CD33 and CD123.
In order to ensure that only the Duo TEAC was purified, one of the chains had a His-tag and the other chain had an EPEA tag. The CD33 dimer and CD33 protein itself would only express the His tag, the CD123 dimer and the CD123 protein itself would only express the EPEA tag when using SEQ ID NOs: 199 and 200. In contrast, the properly assembled Duo-TEAC would express one His tag and one EPEA tag. Therefore, the protein mixture was purified first through a His column and the eluate was then purified through an EPEA column. Thus, the purified protein would only contain the CD33/CD123 Duo-TEAC and not the CD33/CD33 or CD123/CD123 TEACs.
Next, the CD33/CD123 TEACs were evaluated in an in vitro model. An AML tumor cell line, which had previously been confirmed to be CD33+ and CD123+ by flow cytometry, was washed in serum-free media and re-suspended to 105/ml in serum free media. The Dual IgG TEAC or Duo-TEAC was added at varying concentrations between 1-1000 nM and incubated at room temperature for 30 minutes. Excess, unbound TEAC was removed by washing in serum free media, and labelled tumor cells were re-suspended to 106/ml. 100 μl tumor cells were added to triplicate wells of 96 well U-bottom plate. CD3+ T cells were washed twice in serum-free media and re-suspended to 2.5×105/ml. 100 μl T cells were added to each well and the co-culture incubated overnight at 37° C. The following day, supernatant was assayed for the presence of IFN gamma by ELISA (ThermoFisher, USA), and the assay stopped by addition of 10% hydrochloric acid. The plate was read at absorbance of 450 nm in 96 well plate reader (Neo 2, Synergy, USA). Results shown in
A treatment plan could be designed for treating patients by infusing with an anti-CD33/anti-CD123 TEAC (either as a two-component composition or a single-agent). Patients with acute myeloid leukemia (AML) express both CD33 and CD123. Data suggest that an estimated 69.5% of AMLs had simultaneous presence of both antigens (See Ehninger et al., Blood Cancer Journal 4:e218 (2014)). The presence of the half-life extending moiety would be expected to increase the half-life of the molecule in patients to days instead of hours and therefore may allow less-frequent dosing compared to a TEAC without a half-life extension moiety.
A variety of TEACs with half-life extending moieties can be designed with EpCAM targeting moieties.
A two-component Dual IgG TEAC was be designed comprising a first IgG TEAC comprising two targeting moieties that are each an anti-EpCAM antibody and a second IgG TEAC comprising two targeting moieties that are each an anti-EpCAM antibody. Each IgG TEAC also comprises two copies of T-cell engaging domain, two copies of an inert binding partners, and two copies of a protease cleavage site between each T-cell engaging domain and its inert binding partner.
Further, both the first and second components also comprise a linker comprising a half-life extending moiety (SEQ ID NOs: 201 and 202). The half-life extending moiety comprises an Fc domain, wherein one end of the linker is directly attached to the inert binding partner. When expressed in HEK293T cells, one TEAC (EpCAM binding region (scFv)-anti-CD3 domain-cleavage sequence-inert binding partner-Fc domain) will recombine with a second copy of the TEAC via covalent interactions and form the IgG TEAC.
A single-agent TEAC comprising a half-life extending moiety was also designed that comprises two anti-EpCAM targeting moieties, (SEQ ID NOs: 201 and 202).
The first and second inert binding partners of the single-agent TEAC are connected via a linker comprising a half-life extending moiety. The half-life extending moiety comprises an Fc domain, from IgG4 wherein the inert binding partner is directly linked to the Fc domain. When expressed in HEK293T cells, one TEAC (EpCAM binding region (scFv)-anti-CD3 VH domain-cleavage sequence-inert binding partner-Fc domain) will recombine with the second TEAC (containing the same EpCAM binding domain with the corresponding anti-CD3 VL) via covalent interactions and form the Duo IgG TEAC. Both protein chains contain different purification tags (EPEA and histidine) which allows purification of the Duo-TEAC which contains one arm with anti-EpCAMxanti-CD3-VH and the second arm with anti-EpCAMxanti-CD3-VL. The CD3-VH/CD3-VH or CD3-VL/CD3-VL will not be purified.
Constructs were generated and purified in a similar way to the CD33 and CD123 IgG TEAC and CD33/CD123 Duo-TEAC in Example 1. For the IgG TEACs, the EpCAM scFv TEACs was used as a starting construct and the CH domains from an IgG4 antibody sequence were added to the C-terminal end of the inert binding partner. These TEACs were then produced in HEK 293T cells and purified from the supernatant.
For the EpCAM Duo-TEAC, two genes were added into a single plasmid with the scFv sequence of EpCAM TEAC with the IgG4 CH domains. Importantly, each TEAC gene sequence of the Duo-TEAC had a different tag (one had His tag and the other had EPEA tag) to allow purification. Once the proteins had been produced, constructs were run over a His column and then the eluate was run over an EPEA column so that the only protein purified would have both His and EPEA tags and therefore only Duo-TEAC would be used in the assay
A lung cancer tumor cell line (NCI-H2009), which had previously been confirmed to be EpCAM+ by flow cytometry, was washed in serum-free media and re-suspended to 105/ml in serum free media. Anti-EpCAM IgG TEAC or Duo-TEAC was added at varying concentrations between 1-1000 nM and incubated at room temperature for 30 minutes. Excess, unbound TEAC was removed by washing in serum free media, and labelled tumor cells were re-suspended to 106/ml. 100 μl tumor cells were added to triplicate wells of 96 well U-bottom plate. CD3+ T cells were washed twice in serum free media and re-suspended to 2.5×105/ml. 100 μl T cells were added to each well, and the co-culture incubated overnight at 37° C. The following day, supernatant was assayed for the presence of IFN gamma by ELISA (ThermoFisher, USA) and the assay stopped by addition of 10% hydrochloric acid. The plate was read at absorbance of 450 nm in 96 well plate reader (Neo 2, Synergy, USA). Results shown in
A treatment plan could be designed for treating patients by infusing with an anti-EpCAM TEAC (either a two-component composition or a single-agent). Multiple types of solid tumor cancers express EpCAM, including colorectal, prostate, breast and ovarian cancers. In this way the TEACs with half-life extending moieties and EpCAM targeting moieties may have efficacy against a wide range of solid tumors. The presence of the half-life extending moiety would be expected to increase the half-life of the molecule in patients to days instead of hours and therefore may allow less-frequent dosing compared to a TEAC without a half-life extension moiety.
An anti-EpCAM ATTAC (containing the anti-CD3 VH domain) can be designed as an IgG TEAC/ATTAC comprising 2 copies of an anti-EpCAM scFv as targeting moieties, 2 copies of an anti-CD3e VH as immune cell engaging domains, 2 copies of an Ig VL domain as inert binding partners, and MMP2 cleavage sequences between each inert binding partner and immune cell engaging domains.
An anti-CD8 ATTAC (containing the anti-CD3e VL domain) can be designed as an IgG TEAC/ATTAC comprising 2 copies of an anti-CD8 VHH as targeting moieties, 2 copies of an anti-CD3e VL as immune cell engaging domains, 2 copies of an Ig VH domain as inert binding partners, and MMP2 cleavage sequences between each inert binding partner and immune cell engaging domains.
Both the first and second components may comprise a linker comprising a half-life extending moiety. The half-life extending moiety comprises an Fc domain, wherein one end of the linker is directly attached to one copy of the inert binding partner.
Further, a single-agent EpCAM/CD8 ATTAC can be designed with these same moieties. Constructs can be generated and purified in a similar way to the CD33 and CD123 IgG TEAC and CD33/CD123 Duo-TEAC in Example 1. An exemplary single-agent ATTAC could be generated via co-expression of an anti-EpCAM TEAC with a HIS tag (SEQ ID NO: 202) and an anti-CD8 VL ATTAC with an EPEA tag (SEQ ID NO: 212).
Conventional TEACs have been designed to target two different antigens on tumor cells where two antigens would better target tumor cells compared with healthy cells. In tumor cells where a single antigen could be used to target tumor cells, the second targeting moiety could be used to target specific subsets of T cells. In patients with prostate cancer the surface antigen PSMA is a good single target for tumor cells. A treatment plan could be designed for treating patients by infusing patients with EpCAM/CD8 ATTAC (either a two-component composition or a single-agent). In this way the ATTACs with half-life extending moieties may have efficacy against a wide range of solid tumors. The presence of the half-life extending moiety would be expected to increase the half-life of the molecule in patients to days instead of hours and therefore may allow less-frequent dosing compared to a TEAC without a half-life extension moiety.
Half-life extension (HLE) of TEAC or ATTAC molecules could be achieved by various strategies. One strategy is fusion of the TEAC or ATTAC to a protein with long serum half-life, such as the Fc of an antibody or serum albumin. Another strategy is fusion of the TEAC or ATTAC to a binding moiety that binds to a protein with long half-life such as serum albumin.
In principal, this half-life extension by ways of a fusion protein can be done in two opposing ways, which differ in how the TEAC or ATTAC and the half-life extending moiety are fused. First, the fusion protein can be engineered such that the TEAC or ATTAC has extended half-life irrespective of proteolytic activation. Second, the fusion protein can be engineered such that only the prodrug has long half-life, but proteolytic activation of the molecule leads to removal of the half-life extending moiety. The latter can be accomplished by fusing the half-life extending moiety to an inert binding partner of the TEAC or ATTAC.
Quantitative systems pharmacology modelling of TEAC or ATTAC activation has revealed that extending the half-life of the cleaved (active) TEAC or ATTAC could lead to a decreased therapeutic index. This is due to the fact that cleaved molecules could accumulate over time if the rate of cleavage is greater than the rate of clearance of these molecules, and molecules activated by cleavage in the tumor microenvironment could diffuse into circulation and cause off-tumor toxicities.
However, when the half-life extending moiety is fused to an inert binding partner and therefore removed during proteolytic activation of the TEAC or ATTAC, the active compounds have short serum half-live and are quickly eliminated. This prevents accumulation of activated molecules over time and limits unwanted activity of TEACs or ATTACs outside of the tumor microenvironment, where the activation occurs. This is shown in
A TEAC comprising a half-life extension moiety was engineered as an Fc fusion protein using the Fc domain of human IgG1. In this example, the sequence of human IgG1 was modified by mutation of Asparagine 297 to Glutamine (N297Q, EU numbering), which mutates the consensus sequence for an N-linked glycosylation at this site, thereby abrogating glycosylation and producing an aglycosylated Fc domain. Aglycosylated immunoglobulin Fc domains have reduced effector function (Wang X et al. Protein Cell 9(1):63-73 (2018)). The Fc-TEAC molecules were constructed such that the Fc domain is linked to the inert binding partner via a flexible linker, and the FC domain and the inert binding partner are released together following proteolytic activation (
Fc-TEAC fusion proteins were constructed with various linkers between the Fc and the inert binding partner (Table 14). The proteins were expressed in transient HEK293 cultures (30-50 ml) in shake-flasks by co-transfection of the MP251, MP252, MP253, or MP254 constructs (SEQ ID NOs: 175-178, respectively) comprising heavy chains of the targeting moiety with the corresponding light chain construct (MP058, SEQ ID NO: 171) of the targeting moiety to generate TEACs. The expressed proteins were purified from supernatant by FPLC using protein A columns (Mab Select PrismA, GE). Analysis of the purified proteins by SDS-PAGE showed that homogenous products were produced of the expected molecular weight (200 kDa) with all the linker lengths tested in this experiment (
For testing proteolytic cleavage of the Fc-TEAC proteins, the purified proteins were incubated with recombinant Factor Xa (New England Biolabs) at room temperature for 2 hours. The Fc-TEACs were engineered with an MMP2/9 cleavage sequence (SEQ ID NO: 49) in the protease linker, which also contains a cryptic FXa-cleavage site, and thus can be cleaved with recombinant FXa. The digested samples were analyzed by SDS-PAGE, which showed that cleavage produced the expected fragments from the TEACs corresponding to the molecular weight of partially and fully cleaved fragments (
In another example, a two-component TEAC system using the anti-EGFR antibody GA201 (Gerdes and Umana, Clin Cancer Res. 15; 20(4):1055 (2014)) as the targeting moiety was designed, wherein each TEAC component comprises a Fc domain as a half-life extension moiety (i.e. Fc-TEAC components). In this experiment, both components of the two-component TEAC system were constructed using identical Fc-linker and protease linker sequences, as well as the same anti-EGFR antibody GA201 for the targeting moiety.
Two TEAC components were designed, wherein each component comprised the heavy chain of the GA201 antibody. The difference between the two components is that one of components comprises the VH of the CD3 antibody and a corresponding VL domain as an inert binding partner. This component comprising the VH of the CD3 antibody is referred to here as “H-TEAC” or “VH TEAC.” The other component comprises the VL of the CD3 antibody and a corresponding VH domain as an inert binding partner. This component comprising the VL of the CD3 antibody is referred to as “L-TEAC” or “VL TEAC.” The sequence of the H-TEAC heavy chain is SEQ ID NO: 179 (MP268) and the sequence of the L-TEAC heavy chain is SEQ ID NO: 180 (MP269).
To produce the TEAC proteins, these TEACs components comprising the heavy chains of the targeting moiety anti-EGFR antibody GA201 antibody were co-expressed with the light chain MP113 (SEQ ID NO: 174) of GA201. The co-expression produced Fc-TEAC molecules recombinantly in HEK293 cells. The parental unstabilized TEAC molecules lacking the Fc were also produced by transient transfection in HEK293 cells by co-transfection of the heavy chains MP111 (SEQ ID NO: 172) or MP112 (SEQ ID NO: 173) with the light chain MP113 (SEQ ID NO: 174). These TEAC molecules contain a hexa-histidine tag at the C-terminus of the heavy chain to facilitate purification of the proteins by Ni-affinity chromatography. The combination of MP111 (SEQ ID NO: 172) and MP113 (SEQ ID NO: 174) generates construct RO130, while the combination of MP112 (SEQ ID NO: 173) and MP113 (SEQ ID NO: 174) generates construct RO131.
The ability of Fc-TEAC and parental TEAC to bind to EGFR was tested in an ELISA assay. Assay plates were coated with recombinant EGFR ectodomain (2 μg/ml, at 4° C. overnight) and incubated with TEACs at various dilutions. Bound TEAC protein was detected by an HRP-coupled anti-His antibody (Cell Signaling Catalog No. D3I1O).
Results of the EGFR binding ELISA are shown in
The T-cell redirection activity of Fc-TEAC and TEAC constructs was tested in an in vitro T-cell activation assay. Breast cancer cells (MDA-MB-231) were seeded in 96-well plates at a density of 10000 cells/well and allowed to adhere overnight. Peripheral blood mononuclear cells (PBMCs) were added at an effector-to-target ratio of 10:1, and the cells were treated with serial dilutions of TEAC molecules. Secreted interferon gamma was detected in the media 24 hours after addition of TEAC using an IFNgamma ELISA kit (Invitrogen Catalog No. 88-7316-88), and target cell killing was determined 48 hours after the start of treatment using a cytotoxicity assay that quantitatively measures lactate dehydrogenase (LDH) (CytoTox96, Promega). Results of a T cell activation assay (
The serum stability of TEAC and Fc-TEAC molecules was tested in BALB/c mice. Mice were dosed intravenously with 50 μg of Fc-TEAC (RO269) or the corresponding parental TEAC (RO131). Concentration of TEAC in mouse plasma was determined by ELISA using an anti-human Fab capture antibody (Jackson Laboratories #109-005-006) followed by detection with an anti-His secondary antibody coupled to HRP (Cell Signaling Catalog No. D3I1O). The resulting pharmacokinetic profile (
These data confirm that TEAC constructs comprising half-life extension moieties, such as Fc domains, can have improved pharmacokinetic profiles compared to TEAC constructs without half-life-extension moieties.
The foregoing written specification is considered to be sufficient to enable one skilled in the art to practice the embodiments. The foregoing description and Examples detail certain embodiments and describes the best mode contemplated by the inventors. It will be appreciated, however, that no matter how detailed the foregoing may appear in text, the embodiment may be practiced in many ways and should be construed in accordance with the appended claims and any equivalents thereof.
As used herein, the term about refers to a numeric value, including, for example, whole numbers, fractions, and percentages, whether or not explicitly indicated. The term about generally refers to a range of numerical values (e.g., +/−5-10% of the recited range) that one of ordinary skill in the art would consider equivalent to the recited value (e.g., having the same function or result). When terms such as at least and about precede a list of numerical values or ranges, the terms modify all of the values or ranges provided in the list. In some instances, the term about may include numerical values that are rounded to the nearest significant figure.
This application claims the benefit of U.S. Provisional Application No. 62/841,960, filed May 2, 2019. The entire contents of the foregoing are hereby incorporated herein by reference.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2020/029548 | 4/23/2020 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 62841960 | May 2019 | US |
