T cell activators and methods of use thereof

Abstract

The present disclosure relates to multispecific molecules comprising a peptide-MHC complex and an immune cell antigen targeting moiety. Particular embodiments relate to multimeric (e.g., dimeric) molecules comprising a peptide-MHC complex, an immune cell antigen targeting moiety, and a multimerization moiety. The present disclosure further provides pharmaceutical compositions comprising the multispecific molecules, and methods of using the multispecific molecules in antigen-specific T cell activation, in inducing antigen-specific immune responses, and in therapeutic applications, as well as nucleic acids encoding the multispecific molecules, recombinant cells expressing the multispecific molecules, and methods of producing the multispecific molecules.

Description

[Technical Field]

[0001] 1. CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 487,386, filed February 28, 2023, and U.S. Provisional Patent Application No. 63 / 494,604, filed April 6, 2023, the contents of which are incorporated herein by reference in their entireties.

[0002] 2. Sequence Listing This application contains a Sequence Listing that has been submitted electronically in XML format and is incorporated herein by reference in its entirety. The XML Sequence Listing, created on February 28, 2024, is named RGN-033WO_SL.xml and is 229,868 bytes in size. [Background technology]

[0003] T cell activation is important for antigen recognition, which is essential for the immune system to generate an effective response to health threats. Therefore, antigen-specific T cell activation can be used to prevent and / or treat several diseases, such as viral, bacterial, fungal, and / or protozoal infections, as well as various cancers.

[0004] T cell activation is thought to involve three signals: (1) antigen recognition, (2) costimulation, and (3) cytokine-mediated differentiation and expansion. The first signal, often referred to as signal 1, triggers the initial activation of T cells, which usually involves encountering antigenic peptides carried by major histocompatibility complex (MHC) molecules presented on the surface of antigen-presenting cells (APCs). These peptide-MHC (pMHC) molecules bind to the T cell receptor (TCR) complexed with the CD3 coreceptor. Signal 2 involves costimulation of T cells by a secondary signal, such as a molecule that binds to the CD28 coreceptor. This costimulation of T cells regulates their proliferation and survival.

[0005] It has been shown that the treatment of T cells with antibodies against CD3 clusters CD3 on T cells and causes T cell activation in a similar manner to the engagement of TCR by peptide-loaded MHC molecules.However, such activation is not antigen-specific.There is a need in the art for molecules that induce antigen-specific activation and expansion of T cells for the prevention and / or treatment of various infectious diseases and cancers. Summary of the Invention [Means for solving the problem]

[0006] The present disclosure provides multispecific molecules, referred to herein for convenience as "T cell activators," that contain at least one peptide-MHC complex and at least one immune cell antigen targeting moiety (e.g., a T cell antigen targeting moiety, a B cell antigen targeting moiety, etc.). Certain T cell activators described herein include those that contain at least one T cell antigen (TCA) targeting moiety (e.g., a CD3 targeting moiety, a CD28 targeting moiety, etc.), and optionally further contain one or more additional immune cell antigen targeting moieties (e.g., a B cell antigen targeting moiety such as a CD19 targeting moiety, a CD20 targeting moiety, or a CD22 targeting moiety). Without being bound by theory, it is believed that the inclusion of a peptide-MHC complex and a TCA targeting moiety results in a molecule that can cluster and / or activate T cell receptors (TCRs) specific for a particular antigen (e.g., a viral antigen, a tumor antigen, etc.) independent of an antigen-presenting cell. As further described herein, when the TCA targeting moiety is a CD3 targeting moiety, certain disclosed T cell activators can activate TCR signaling independently of costimulatory signal 2. Additional T cell activators described herein include those that comprise at least one B cell antigen (BCA) targeting moiety (e.g., a CD19 targeting moiety, a CD20 targeting moiety, a CD22 targeting moiety, etc.), and optionally further comprise one or more additional immune cell antigen targeting moieties (e.g., a T cell antigen targeting moiety such as a CD28 targeting moiety or a CD3 targeting moiety). The T cell activators described herein are useful in a variety of therapeutic and prophylactic methods in which stimulation of the host's immune system is beneficial.

[0007] Peptide-MHC complexes that can be used in the T cell activators of the present disclosure are described in Section 6.3. Immune cell antigen targeting moieties that can be used in the T cell activators of the present disclosure are described in Section 6.4.

[0008] In certain cases, the T cell activator also includes a tumor antigen targeting moiety. Tumor antigen targeting moieties that can be used in the T cell activators of the present disclosure are described in Section 6.5.

[0009] In some cases, the T cell activators also include a multimerizing moiety, such as an Fc domain. Multimerizing moieties that can be used in the T cell activators of the present disclosure are described in Section 6.7.

[0010] Various exemplary configurations of the T cell activators of the present disclosure are described in specific embodiments 1-566 below. Linkers that can be used to connect different components of the T cell activators of the present disclosure are described in Section 6.8.

[0011] The present disclosure further provides nucleic acids encoding the T cell activators of the present disclosure. The nucleic acids encoding the T cell activators can be a single nucleic acid (e.g., a vector encoding all polypeptide chains of the T cell activator) or multiple nucleic acids (e.g., two or more vectors encoding different polypeptide chains of the T cell activator). The present disclosure further provides host cells and cell lines engineered to express the nucleic acids and T cell activators of the present disclosure. The present disclosure further provides methods of producing the T cell activators of the present disclosure. Exemplary nucleic acids, host cells, and cell lines, as well as methods of producing the T cell activators, are described in Section 6.9, below, and in specific embodiments 567-570.

[0012] The present disclosure further provides pharmaceutical compositions comprising a T cell activator of the present disclosure (or a nucleic acid encoding a T cell activator), optionally in addition to one or more multispecific antigen-binding molecules (e.g., as described in Section 6.6). Exemplary pharmaceutical compositions are described in Section 6.10, below, and in specific embodiments 575-580, with exemplary pharmaceutical compositions comprising a T cell activator polypeptide described in Section 6.10.1 and exemplary pharmaceutical compositions comprising a T cell activator encoding nucleic acid described in Section 6.10.2.

[0013] Further provided herein are methods of using the T cell activators and pharmaceutical compositions of the present disclosure, for example, to treat or prevent cancer. Exemplary methods are described in Section 6.11 and include therapeutic methods (e.g., via delivery of a T cell activator polypeptide) and prophylactic methods (e.g., via delivery of a nucleic acid encoding the T cell activator, such as a plasmid, DNA, mRNA, or viral vector). The T cell activators of the present disclosure are useful in combination therapies, for example, in combination with multispecific antigen binding molecules (e.g., as described in Section 6.6), cytokine therapeutics, or other cancer therapeutics. Exemplary combination therapies are disclosed in Section 6.12. Specific embodiments of the disclosed therapeutic, prophylactic, and immune cell activation methods are described below in specific embodiments 581-637. [Brief explanation of the drawings]

[0014] [Figure 1A]1 shows an exemplary T cell activator construct of the present disclosure. (1) represents the Fc domain of an immunoglobulin, e.g., an IgG such as IgG1 or IgG4, as described in Section 6.7.1, which typically includes a CH2 domain, a CH3 domain, and optionally a hinge region (separately represented as (2) in Figure 1), as described, e.g., in Section 6.7.1.3. (2) represents the hinge region of an immunoglobulin Fc domain, e.g., an IgG hinge domain, which in various embodiments is an IgG1 or IgG4 hinge domain or a chimeric hinge domain as described in Section 6.7.1.3. (3) represents an immune cell antigen targeting moiety (shown as a Fab domain, but can be any immune cell antigen targeting moiety described herein), e.g., a T cell targeting moiety (e.g., the antigen-binding domain of an anti-CD3 or anti-CD28 antibody) or a B cell targeting moiety (e.g., the antigen-binding domain of an anti-CD19, anti-CD20, or anti-CD22 antibody), as described in Section 6.4. (4) represents the peptide-MHC (pMHC) complex described in Section 6.3. (5-6) represents a linker, such as those described in Section 6.8. A linker can be used to connect the pMHC complex to the hinge region at the N-terminus of the Fc domain, or to connect an immune cell antigen targeting moiety or a tumor antigen targeting moiety to the C-terminus of the Fc domain. (5) represents the linker between the pMHC complex and the hinge region at the N-terminus of the Fc domain. (6) represents the linker between the C-terminus of the Fc domain and the targeting moiety. (7) represents the tumor antigen targeting moiety (shown as a Fab domain, but may be any tumor antigen targeting moiety). Asymmetric constructs are shown as Fc heterodimers with knob (bulge) and hole (cavity) mutations to promote heterodimerization and "star" (star) mutations to facilitate purification. However, any alternative heterodimerization strategy may be used, or the knob and hole and / or star mutation chains may be reversed. [Figure 1B] Same as above. [Figure 1C] Same as above. [Figure 1D] Same as above. [Figure 1E] Same as above. [Figure 1F] Same as above. [Figure 1G] Same as above. [Figure 1H] Same as above. [Figure 1I] Same as above. [Figure 2A] Figure 1 shows activation of Jurkat reporter cells by plate-bound monovalent T cell activator constructs, each containing a single pMHC complex. Figure 2 shows activation of Jurkat / NFAT-Luc / Cl 3C7 / HPV16E7(11-19)TCR709 cells by plate-bound T cell activator constructs, each containing a single pMHC complex, with each pMHC complex connected to the hinge region of the Fc domain via a short linker. [Figure 2B] Figure 1 shows activation of Jurkat reporter cells by plate-bound monovalent T cell activator constructs, each containing a single pMHC complex. Figure 2 shows activation of Jurkat / NFAT-Luc / Cl 3C7 / HPV16E6(29-38) TCR cells by plate-bound T cell activator constructs, in which the pMHC complexes are connected to the hinge region of the Fc domain via a short linker. [Figure 2C] Figure 1 shows activation of Jurkat reporter cells by plate-bound monovalent T cell activator constructs, each containing a single pMHC complex. Figure 2 shows activation of Jurkat / NFAT-Luc / Cl 3C7 / HPV16E7(11-19)TCR709 cells by plate-bound T cell activator constructs, in which the pMHC complexes are connected to the hinge region of the Fc domain via a long linker. [Figure 2D] Figure 1 shows activation of Jurkat reporter cells by plate-bound monovalent T cell activator constructs, each containing a single pMHC complex. Figure 2 shows activation of Jurkat / NFAT-Luc / Cl 3C7 / HPV16E6(29-38) TCR cells by plate-bound T cell activator constructs, in which the pMHC complexes are connected to the hinge region of the Fc domain via a long linker. [Figure 3A]Figure 1 shows activation of Jurkat reporter cells by soluble monovalent T cell activator constructs, each containing a single pMHC complex. Figure 2 shows activation of Jurkat / NFAT-Luc / Cl 3C7 / HPV16E7(11-19)TCR709 cells by soluble T cell activator constructs, in which the pMHC complex is connected to the hinge region of the Fc domain via a short linker. [Figure 3B] Figure 1 shows activation of Jurkat reporter cells by soluble monovalent T cell activator constructs, each containing a single pMHC complex. Figure 2 shows activation of Jurkat / NFAT-Luc / Cl 3C7 / HPV16E6(29-38) TCR cells by soluble T cell activator constructs, in which the pMHC complex is connected to the hinge region of the Fc domain via a short linker. [Figure 3C] Figure 1 shows activation of Jurkat reporter cells by soluble monovalent T cell activator constructs, each containing a single pMHC complex. Figure 2 shows activation of Jurkat / NFAT-Luc / Cl 3C7 / HPV16E7(11-19)TCR709 cells by soluble T cell activator constructs, in which the pMHC complex is connected to the hinge region of the Fc domain via a long linker. [Figure 3D] Figure 1 shows activation of Jurkat reporter cells by soluble monovalent T cell activator constructs, each containing a single pMHC complex. Figure 2 shows activation of Jurkat / NFAT-Luc / Cl 3C7 / HPV16E6(29-38) TCR cells by soluble T cell activator constructs, in which the pMHC complex is connected to the hinge region of the Fc domain via a long linker. [Figure 4A] Figure 1 shows activation of Jurkat reporter cells by plate-bound bivalent T cell activator constructs, each containing two pMHC complexes at the N-terminus of the Fc domain. Figure 2 shows activation of Jurkat / NFAT-Luc / Cl 3C7 / HPV16E7(11-19)TCR709 cells by plate-bound T cell activator constructs, in which the pMHC complexes are connected to the hinge region of the Fc domain via a short linker. [Figure 4B]Figure 1 shows activation of Jurkat reporter cells by plate-bound bivalent T cell activator constructs, each containing two pMHC complexes at the N-terminus of the Fc domain. Figure 2 shows activation of Jurkat / NFAT-Luc / Cl 3C7 / HPV16E6(29-38) TCR cells by plate-bound T cell activator constructs, in which the pMHC complexes are connected to the hinge region of the Fc domain via a short linker. [Figure 4C] Figure 1 shows activation of Jurkat reporter cells by plate-bound bivalent T cell activator constructs, each containing two pMHC complexes at the N-terminus of the Fc domain. Figure 2 shows activation of Jurkat / NFAT-Luc / Cl 3C7 / HPV16E7(11-19)TCR709 cells by plate-bound T cell activator constructs, in which the pMHC complexes are connected to the hinge region of the Fc domain via a long linker. [Figure 4D] Figure 1 shows activation of Jurkat reporter cells by plate-bound bivalent T cell activator constructs, each containing two pMHC complexes at the N-terminus of the Fc domain. Figure 2 shows activation of Jurkat / NFAT-Luc / Cl 3C7 / HPV16E6(29-38) TCR cells by plate-bound T cell activator constructs, in which the pMHC complexes are connected to the hinge region of the Fc domain via a long linker. [Figure 5A] Figure 1 shows activation of Jurkat reporter cells by soluble bivalent T cell activator constructs, each containing two pMHC complexes at the N-terminus of the Fc domain. Figure 2 shows activation of Jurkat / NFAT-Luc / Cl 3C7 / HPV16E7(11-19)TCR709 cells by soluble T cell activator constructs, in which the pMHC complexes are connected to the hinge region of the Fc domain via a short linker. [Figure 5B] Figure 1 shows activation of Jurkat reporter cells by soluble bivalent T cell activator constructs, each containing two pMHC complexes at the N-terminus of the Fc domain. Figure 2 shows activation of Jurkat / NFAT-Luc / Cl 3C7 / HPV16E6(29-38) TCR cells by soluble T cell activator constructs, in which the pMHC complexes are connected to the hinge region of the Fc domain via a short linker. [Figure 5C] Figure 1 shows activation of Jurkat reporter cells by soluble bivalent T cell activator constructs, each containing two pMHC complexes at the N-terminus of the Fc domain. Figure 2 shows activation of Jurkat / NFAT-Luc / Cl 3C7 / HPV16E7(11-19)TCR709 cells by plate-bound T cell activator constructs, in which the pMHC complexes are connected to the hinge region of the Fc domain via a long linker. [Figure 5D] Figure 1 shows activation of Jurkat reporter cells by soluble bivalent T cell activator constructs, each containing two pMHC complexes at the N-terminus of the Fc domain. Figure 2 shows activation of Jurkat / NFAT-Luc / Cl 3C7 / HPV16E6(29-38) TCR cells by plate-bound T cell activator constructs, in which the pMHC complexes are connected to the hinge region of the Fc domain via a long linker. [Figure 6A] Figure 1 shows the effect of pre-clustering bivalent T cell activators with anti-Fc on Jurkat reporter cell activation. Activation of Jurkat / NFAT-Luc / Cl 3C7 / HPV16E7(11-19)TCR709 cells by pre-clustered T cell activator constructs is shown, in which the pMHC complex is connected to the hinge region of the Fc domain via a short linker. [Figure 6B] Figure 1 shows the effect of pre-clustering bivalent T cell activators with anti-Fc on Jurkat reporter cell activation. Activation of Jurkat / NFAT-Luc / Cl 3C7 / HPV16E6(29-38) TCR cells by pre-clustered T cell activator constructs is shown, in which the pMHC complex is connected to the hinge region of the Fc domain via a long linker. [Figure 6C] Figure 1 shows the effect of pre-clustering bivalent T cell activators with anti-Fc on Jurkat reporter cell activation. Activation of Jurkat / NFAT-Luc / Cl 3C7 / HPV16E7(11-19)TCR709 cells by pre-clustered T cell activator constructs is shown, in which the pMHC complex is connected to the hinge region of the Fc domain via a long linker. [Figure 6D]Figure 1 shows the effect of pre-clustering bivalent T cell activators with anti-Fc on Jurkat reporter cell activation. Activation of Jurkat / NFAT-Luc / Cl 3C7 / HPV16E6(29-38) TCR cells by pre-clustered T cell activator constructs is shown, in which the pMHC complex is connected to the hinge region of the Fc domain via a long linker. [Figure 7A] 1 shows IFN-γ release by primary TCR-T cells upon treatment with a monovalent T cell activator of the present disclosure. 1 shows IFN-γ release in MAGE-A4 PN45545(230-239) TCR-T cells upon treatment with a monovalent T cell activator, where each pMHC complex is connected to the hinge region of the Fc domain via a short linker. [Figure 7B] Figure 1 shows IFN-γ release by primary TCR-T cells upon treatment with a monovalent T cell activator of the present disclosure. Figure 2 shows IFN-γ release in control HPVE6 TCR-T cells upon treatment with a monovalent T cell activator, where each pMHC complex is connected to the hinge region of the Fc domain via a short linker. [Figure 7C] 1 shows IFN-γ release by primary TCR-T cells upon treatment with a monovalent T cell activator of the present disclosure. 1 shows IFN-γ release in MAGE-A4 PN45545(230-239) TCR-T cells upon treatment with a monovalent T cell activator, where each pMHC complex is connected to the hinge region of the Fc domain via a long linker. [Figure 7D] 1 shows IFN-γ release by primary TCR-T cells upon treatment with a monovalent T cell activator of the present disclosure. 1 shows IFN-γ release in HPV E6 TCR-T negative control cells upon treatment with a monovalent T cell activator, where each pMHC complex is connected to the hinge region of the Fc domain via a long linker. [Figure 7E] 1 shows IFN-γ release by primary TCR-T cells upon treatment with a monovalent T cell activator of the present disclosure. 1 shows IFN-γ release in HPV16E7 TCR709 TCR-T cells upon treatment with a monovalent T cell activator, where each pMHC complex is connected to the hinge region of the Fc domain via a short linker. [Figure 7F] 1 shows IFN-γ release by primary TCR-T cells upon treatment with a monovalent T cell activator of the present disclosure. 1 shows IFN-γ release in NY-ESO-1 1G4 TCR-T negative control cells upon treatment with a monovalent T cell activator, where each pMHC complex is connected to the hinge region of the Fc domain via a short linker. [Figure 7G] 1 shows IFN-γ release by primary TCR-T cells upon treatment with a monovalent T cell activator of the present disclosure. 1 shows IFN-γ release in HPV16E7 TCR709 TCR-T cells upon treatment with a monovalent T cell activator, where each pMHC complex is connected to the hinge region of the Fc domain via a long linker. [Figure 7H] 1 shows IFN-γ release by primary TCR-T cells upon treatment with a monovalent T cell activator of the present disclosure. 1 shows IFN-γ release in NY-ESO-1 1G4 TCR-T negative control cells upon treatment with a monovalent T cell activator, where each pMHC complex is connected to the hinge region of the Fc domain via a long linker. [Figure 8A] 1 shows the tumor cell-killing activity of TCR-T cells primed with T cell activators containing HPV16E7 pMHC complexes. This is a table showing the primed target TCR-T cells and anti-CD3 partial FACS EC50 values ​​for each T cell activator. [Figure 8B] Figure 1 shows the tumor cell killing activity of TCR-T cells primed with T cell activators containing HPV16E7 pMHC complexes. Maximum percent lysis of CaSki tumor cells as a function of T cell:tumor cell. [Figure 9A] Figure 1 shows that reporter cell activation is related to cis-binding of T cell activators. Figure 1 shows HPV16E7 TCR709 NFAT luciferase reporter cell activity upon application of T cell activators containing NY-ESO-1(157-165) pMHC and a short linker to mixed Jurkat cells. [Figure 9B]Figure 1 shows that reporter cell activation is related to cis-binding of T cell activators. Figure 1 shows HPV16E7 TCR709 NFAT luciferase reporter cell activity upon application of T cell activators containing NY-ESO-1(157-165) pMHC and a long linker to mixed Jurkat cells. [Figure 9C] Figure 1 shows that reporter cell activation is related to cis-binding of T cell activators. Positive controls show HPV16E7 TCR709 NFAT luciferase reporter cell activity upon application of HPV E7(11-19) pMHC and T cell activators containing either short or long linkers, respectively. [Figure 9D] Figure 1 shows that reporter cell activation is related to cis-binding of T cell activators. Positive controls show HPV16E7 TCR709 NFAT luciferase reporter cell activity upon application of HPV E7(11-19) pMHC and T cell activators containing either short or long linkers, respectively. [Figure 10A] Figure 1 shows histograms showing antigen-specific expansion of CMVpp65 T cells upon treatment with low concentrations of T cell activators containing long linkers. CMVpp65+ T cells are represented by the light-shaded curve, CMVpp65- CD8+ T cells are represented by the dark-shaded curve, and non-expanded CMV PBMCs (sham control) are represented by the dotted curve. CMVpp65-peptide-pulsed cell populations are shown. [Figure 10B] This histogram shows antigen-specific expansion of CMVpp65 T cells upon treatment with low concentrations of T cell activators containing long linkers. CMVpp65+ T cells are represented by the light-shaded curve, CMVpp65-CD8+ T cells are represented by the dark-shaded curve, and non-expanded CMV PBMCs (sham control) are represented by the dotted curve. Cell populations treated with 0.2 nM CMVpp65 pMHCxCD3(7221G20) T cell activators containing long linkers are shown. [Figure 10C]Histogram showing antigen-specific expansion of CMVpp65 T cells upon treatment with low concentrations of T cell activators containing long linkers. CMVpp65+ T cells are represented by the light-shaded curve, CMVpp65-CD8+ T cells are represented by the dark-shaded curve, and non-expanded CMV PBMCs (sham control) are represented by the dotted curve. Cell populations treated with 5 nM CMVpp65 pMHCxCD3(7221G20) T cell activators containing long linkers are shown. [Figure 10D] This histogram shows the antigen-specific expansion of CMVpp65 T cells upon treatment with low concentrations of T cell activators containing long linkers. CMVpp65+ T cells are represented by the light-shaded curve, CMVpp65-CD8+ T cells are represented by the dark-shaded curve, and non-expanded CMV PBMCs (sham control) are represented by the dotted curve. Cell populations treated with 0.2 nM MAGE-A4 pMHCxCD3 (7221G20) containing long linkers are shown. [Figure 10E] This histogram shows antigen-specific expansion of CMVpp65 T cells upon treatment with low concentrations of T cell activators containing long linkers. CMVpp65+ T cells are represented by the light-shaded curve, CMVpp65-CD8+ T cells are represented by the dark-shaded curve, and non-expanded CMV PBMCs (sham control) are represented by the dotted curve. Cell populations treated with 5 nM MAGE-A4 pMHCxCD3 containing long linkers are shown. [Figure 10F] Figure 1 shows histograms showing antigen-specific expansion of CMVpp65 T cells upon treatment with low concentrations of T cell activators containing long linkers. CMVpp65+ T cells are represented by the light-shaded curve, CMVpp65-CD8+ T cells are represented by the dark-shaded curve, and non-expanded CMV PBMCs (sham control) are represented by the dotted curve. The percentage of proliferation in tetramer+ and tetramer- cells upon treatment with CMVpp65 pMHCxCD3(7221G20) T cell activators containing long linkers is shown. [Figure 11A]Figure 1 shows histograms showing antigen-specific expansion of CMVpp65 T cells upon treatment with low concentrations of T cell activators containing short linkers. CMVpp65+ T cells are represented by the light-shaded curve, CMVpp65- CD8+ T cells are represented by the dark-shaded curve, and non-expanded CMV PBMCs (sham control) are represented by the dotted curve. CMVpp65-peptide-pulsed cell populations are shown. [Figure 11B] This histogram shows antigen-specific expansion of CMVpp65 T cells upon treatment with low concentrations of T cell activators containing short linkers. CMVpp65+ T cells are represented by the light-shaded curve, CMVpp65-CD8+ T cells are represented by the dark-shaded curve, and non-expanded CMV PBMCs (sham control) are represented by the dotted curve. Cell populations treated with 0.2 nM CMVpp65 pMHCxCD3 (7221G20) T cell activators containing short linkers are shown. [Figure 11C] Figure 1 shows histograms showing antigen-specific expansion of CMVpp65 T cells upon treatment with low concentrations of T cell activators containing short linkers. CMVpp65+ T cells are represented by the light-shaded curve, CMVpp65-CD8+ T cells are represented by the dark-shaded curve, and non-expanded CMV PBMCs (sham control) are represented by the dotted curve. Cell populations treated with 5 nM CMVpp65 pMHCxCD3 (7221G20) T cell activators containing short linkers are shown. [Figure 11D] This histogram shows antigen-specific expansion of CMVpp65 T cells upon treatment with low concentrations of T cell activators containing short linkers. CMVpp65+ T cells are represented by the light-shaded curve, CMVpp65-CD8+ T cells are represented by the dark-shaded curve, and non-expanded CMV PBMCs (sham control) are represented by the dotted curve. Cell populations treated with 0.2 nM MAGE-A4 pMHCxCD3 (7221G20) containing short linkers are shown. [Figure 11E]This histogram shows the antigen-specific expansion of CMVpp65 T cells upon treatment with low concentrations of T cell activators containing short linkers. CMVpp65+ T cells are represented by the light-shaded curve, CMVpp65-CD8+ T cells are represented by the dark-shaded curve, and non-expanded CMV PBMCs (sham control) are represented by the dotted curve. Cell populations treated with 5 nM MAGE-A4 pMHCxCD3 (7221G20) containing short linkers are shown. [Figure 11F] Figure 1 shows histograms showing antigen-specific expansion of CMVpp65 T cells upon treatment with low concentrations of T cell activators containing short linkers. CMVpp65+ T cells are represented by the light-shaded curve, CMVpp65-CD8+ T cells are represented by the dark-shaded curve, and non-expanded CMV PBMCs (sham control) are represented by the dotted curve. The percentage of proliferation in tetramer+ and tetramer- cells upon treatment with CMVpp65 pMHCxCD3 (7221G20) T cell activators containing short linkers is shown. [Figure 12A] Figure 1 shows antigen-specific expansion of CMVpp65 T cells upon treatment with low concentrations of T cell activators containing CMVpp65 pMHC, shown as the frequency of T cells as determined by flow cytometry, and the frequency of CD8+ tetramer+ and TCRα / β+ tetramer+ cells, respectively, in non-expanded CMV PBMCs. [Figure 12B] Figure 1 shows antigen-specific expansion of CMVpp65 T cells upon treatment with low concentrations of T cell activators containing CMVpp65 pMHC, shown as the frequency of T cells as determined by flow cytometry, and the frequency of CD8+ tetramer+ and TCRα / β+ tetramer+ cells, respectively, in non-expanded CMV PBMCs. [Figure 12C] Figure 1 shows the antigen-specific expansion of CMVpp65 T cells upon treatment with low concentrations of T cell activators containing CMVpp65 pMHC, as determined by flow cytometry. The figures show the frequencies of CD8+ tetramer+ and TCRα / β+ tetramer+ cells, respectively, in CMVpp65 peptide-pulsed CMVpp65+ T cells. [Figure 12D]Figure 1 shows the antigen-specific expansion of CMVpp65 T cells upon treatment with low concentrations of T cell activators containing CMVpp65 pMHC, as determined by flow cytometry. The figures show the frequencies of CD8+ tetramer+ and TCRα / β+ tetramer+ cells, respectively, in CMVpp65 peptide-pulsed CMVpp65+ T cells. [Figure 12E] Figure 1 shows the antigen-specific expansion of CMVpp65 T cells upon treatment with low concentrations of T cell activators containing CMVpp65 pMHC, as determined by flow cytometry, as well as the frequency of CD8+ tetramer+ and TCRα / β+ tetramer+ cells in CMVpp65 cells treated with 0.2 nM CMVpp65 pMHC×CD3(7221G20) T cell activator containing a long linker, respectively. [Figure 12F] Figure 1 shows the antigen-specific expansion of CMVpp65 T cells upon treatment with low concentrations of T cell activators containing CMVpp65 pMHC, as determined by flow cytometry, as well as the frequency of CD8+ tetramer+ and TCRα / β+ tetramer+ cells in CMVpp65 cells treated with 0.2 nM CMVpp65 pMHC×CD3(7221G20) T cell activator containing a long linker, respectively. [Figure 12G] Figure 1 shows the antigen-specific expansion of CMVpp65 T cells upon treatment with low concentrations of T cell activators containing CMVpp65 pMHC, as determined by flow cytometry, as well as the frequency of CD8+ tetramer+ and TCRα / β+ tetramer+ cells in CMVpp65 cells treated with 5 nM of CMVpp65 pMHC×CD3(7221G20) T cell activator containing a long linker, respectively. [Figure 12H]Figure 1 shows the antigen-specific expansion of CMVpp65 T cells upon treatment with low concentrations of T cell activators containing CMVpp65 pMHC, as determined by flow cytometry, as well as the frequency of CD8+ tetramer+ and TCRα / β+ tetramer+ cells in CMVpp65 cells treated with 5 nM of CMVpp65 pMHC×CD3(7221G20) T cell activator containing a long linker, respectively. [Figure 12I] Figure 1 shows the antigen-specific expansion of CMVpp65 T cells upon treatment with low concentrations of T cell activators containing CMVpp65 pMHC, as determined by flow cytometry, and the frequency of CD8+ tetramer+ and TCRα / β+ tetramer+ cells in CMVpp65 cells treated with 0.2 nM MAGE-A4 pMHC×CD3 (7221G20) control T cell activator containing a long linker, respectively. [Figure 12J] Figure 1 shows the antigen-specific expansion of CMVpp65 T cells upon treatment with low concentrations of T cell activators containing CMVpp65 pMHC, as determined by flow cytometry, and the frequency of CD8+ tetramer+ and TCRα / β+ tetramer+ cells in CMVpp65 cells treated with 0.2 nM MAGE-A4 pMHC×CD3 (7221G20) control T cell activator containing a long linker, respectively. [Figure 12K] Figure 1 shows the antigen-specific expansion of CMVpp65 T cells upon treatment with low concentrations of T cell activators containing CMVpp65 pMHC, as determined by flow cytometry, and the frequency of CD8+ tetramer+ and TCRα / β+ tetramer+ cells in CMVpp65 cells treated with 5 nM MAGE-A4 pMHC×CD3 (7221G20) control T cell activator containing a long linker, respectively. [Figure 12L]Figure 1 shows the antigen-specific expansion of CMVpp65 T cells upon treatment with low concentrations of T cell activators containing CMVpp65 pMHC, as determined by flow cytometry, and the frequency of CD8+ tetramer+ and TCRα / β+ tetramer+ cells in CMVpp65 cells treated with 5 nM MAGE-A4 pMHC×CD3 (7221G20) control T cell activator containing a long linker, respectively. [Figure 12M] Figure 1 shows the antigen-specific expansion of CMVpp65 T cells upon treatment with low concentrations of T cell activators containing CMVpp65 pMHC, as determined by flow cytometry, and the frequency of CD8+ tetramer+ and TCRα / β+ tetramer+ cells in CMVpp65 cells treated with 0.2 nM CMVpp65 pMHC×CD3(7221G20) T cell activator containing a short linker, respectively. [Figure 12N] Figure 1 shows the antigen-specific expansion of CMVpp65 T cells upon treatment with low concentrations of T cell activators containing CMVpp65 pMHC, as determined by flow cytometry, and the frequency of CD8+ tetramer+ and TCRα / β+ tetramer+ cells in CMVpp65 cells treated with 0.2 nM CMVpp65 pMHC×CD3(7221G20) T cell activator containing a short linker, respectively. [Figure 12O] Figure 1 shows the antigen-specific expansion of CMVpp65 T cells upon treatment with low concentrations of T cell activators containing CMVpp65 pMHC, as determined by flow cytometry, as well as the frequency of CD8+ tetramer+ and TCRα / β+ tetramer+ cells in CMVpp65 cells treated with 5 nM of CMVpp65 pMHC×CD3(7221G20) T cell activator containing a short linker, respectively. [Figure 12P]Figure 1 shows the antigen-specific expansion of CMVpp65 T cells upon treatment with low concentrations of T cell activators containing CMVpp65 pMHC, as determined by flow cytometry, as well as the frequency of CD8+ tetramer+ and TCRα / β+ tetramer+ cells in CMVpp65 cells treated with 5 nM of CMVpp65 pMHC×CD3(7221G20) T cell activator containing a short linker, respectively. [Figure 12Q] Figure 1 shows the antigen-specific expansion of CMVpp65 T cells upon treatment with low concentrations of T cell activators containing CMVpp65 pMHC, as determined by flow cytometry, and the frequency of CD8+ tetramer+ and TCRα / β+ tetramer+ cells in CMVpp65 cells treated with 0.2 nM MAGE-A4 pMHC×CD3 (7221G20) control T cell activator containing a short linker, respectively. [Figure 12R] Figure 1 shows the antigen-specific expansion of CMVpp65 T cells upon treatment with low concentrations of T cell activators containing CMVpp65 pMHC, as determined by flow cytometry, and the frequency of CD8+ tetramer+ and TCRα / β+ tetramer+ cells in CMVpp65 cells treated with 0.2 nM MAGE-A4 pMHC×CD3 (7221G20) control T cell activator containing a short linker, respectively. [Figure 12S] Figure 1 shows the antigen-specific expansion of CMVpp65 T cells upon treatment with low concentrations of T cell activators containing CMVpp65 pMHC, as determined by flow cytometry, and the frequency of CD8+ tetramer+ and TCRα / β+ tetramer+ cells in CMVpp65 cells treated with 5 nM MAGE-A4 pMHC×CD3 (7221G20) control T cell activator containing a short linker, respectively. [Figure 12T]Figure 1 shows the antigen-specific expansion of CMVpp65 T cells upon treatment with low concentrations of T cell activators containing CMVpp65 pMHC, as determined by flow cytometry, and the frequency of CD8+ tetramer+ and TCRα / β+ tetramer+ cells in CMVpp65 cells treated with 5 nM MAGE-A4 pMHC×CD3 (7221G20) control T cell activator containing a short linker, respectively. [Figure 13A] Figure 1 shows the effect of CD22 × CD28 costimulation on antigen-specific T cell activation by immobilized T cell activators. Results are shown from a mixture of Jurkat / NFAT-Luc / Cl 3C7 / HPV16E7(11-19)TCR709 cells and Raji dKO (CD80 CD86 KO) cells with a T cell activator construct, in which the pMHC complex was connected to the hinge region of the Fc domain via a short linker either costimulated with 31.25 μg / ml of CD22 × CD28 (solid line) or without costimulation (dashed line). [Figure 13B] Figure 1 shows the effect of CD22xCD28 costimulation on antigen-specific T cell activation by immobilized T cell activators. Results are shown from a mixture of HPV16E6(29-38) TCR Jurkat cells and Raji dKO (CD80 CD86 KO) cells with a T cell activator construct, in which the pMHC complex is connected to the hinge region of the Fc domain via a short linker either costimulated with 31.25 μg / ml of CD22xCD28 (solid line) or without costimulation (dashed line). [Figure 13C] Figure 1 shows the effect of CD22 × CD28 costimulation on antigen-specific T cell activation by immobilized T cell activators. Results are shown from a mixture of Jurkat / NFAT-Luc / Cl 3C7 / HPV16E7(11-19)TCR709 cells and Raji dKO (CD80 CD86 KO) cells with a T cell activator construct, in which the pMHC complex was connected to the hinge region of the Fc domain via a short linker either costimulated with 62.5 μg / ml of CD22 × CD28 (solid line) or without costimulation (dashed line). [Figure 13D]Figure 1 shows the effect of CD22xCD28 costimulation on antigen-specific T cell activation by immobilized T cell activators. Results are shown from a mixture of HPV16E6(29-38) TCR Jurkat cells and Raji dKO (CD80 CD86 KO) cells with a T cell activator construct, in which the pMHC complex is connected to the hinge region of the Fc domain via a short linker either costimulated with 62.5 μg / ml of CD22xCD28 (solid line) or without costimulation (dashed line). [Figure 13E] Figure 1 shows the effect of CD22 × CD28 costimulation on antigen-specific T cell activation by immobilized T cell activators. Results are shown from a mixture of Jurkat / NFAT-Luc / Cl 3C7 / HPV16E7(11-19)TCR709 cells and Raji dKO (CD80 CD86 KO) cells with a T cell activator construct, in which the pMHC complex is connected to the hinge region of the Fc domain via a short linker either costimulated with 125 μg / ml of CD22 × CD28 (solid line) or without costimulation (dashed line). [Figure 13F] Figure 1 shows the effect of CD22xCD28 costimulation on antigen-specific T cell activation by immobilized T cell activators. Results are shown from a mixture of HPV16E6(29-38) TCR Jurkat cells and Raji dKO (CD80 CD86 KO) cells with a T cell activator construct, in which the pMHC complex is connected to the hinge region of the Fc domain via a short linker either costimulated with 125 μg / ml of CD22xCD28 (solid line) or without costimulation (dashed line). [Figure 14A] Figure 1 shows the effect of CD22xCD28 costimulation on antigen-specific T cell activation by immobilized pMHCxCD20 T cell activator. Results are shown from a mixture of Jurkat / NFAT-Luc / Cl 3C7 / HPV16E7(11-19) TCR cells and Raji dKO (CD80 CD86 KO) cells co-stimulated with a pMHCxCD20 T cell activator construct, where the pMHC complex is connected to the hinge region of the Fc domain via a short linker, followed by costimulation with various concentrations of CD22xCD28. [Figure 14B] Figure 1 shows the effect of CD22xCD28 costimulation on antigen-specific T cell activation by immobilized pMHCxCD20 T cell activator. Results are shown from a mixture of HPV16E6(29-38) TCR Jurkat cells and Raji dKO (CD80 CD86 KO) cells co-stimulated with a pMHCxCD20 T cell activator construct, where the pMHC complex is connected to the hinge region of the Fc domain via a short linker, followed by co-stimulation with various concentrations of CD22xCD28. [Figure 14C] Figure 1 shows the effect of CD22xCD28 costimulation on antigen-specific T cell activation by immobilized pMHCxCD20 T cell activator. Results are shown from a mixture of Jurkat / NFAT-Luc / Cl 3C7 / HPV16E7(11-19)TCR709 cells and Raji dKO (CD80 CD86 KO) cells co-stimulated with the pMHCxCD20 T cell activator construct, where the pMHC complex is connected to the hinge region of the Fc domain via a long linker. The cells were co-stimulated with various concentrations of CD22xCD28. [Figure 14D] Figure 1 shows the effect of CD22xCD28 costimulation on antigen-specific T cell activation by immobilized pMHCxCD20 T cell activator. Results are shown from a mixture of HPV16E6(29-38) TCR Jurkat cells and Raji dKO (CD80 CD86 KO) cells co-stimulated with the pMHCxCD20 T cell activator construct, where the pMHC complex is connected to the hinge region of the Fc domain via a long linker, followed by co-stimulation with various concentrations of CD22xCD28. [Figure 15A] Figure 1 shows activation of Jurkat reporter cells by T cell activator constructs with IgG1 or IgG4 Fc domains, where each pMHC complex is connected to the hinge region of the IgG1 or IgG4 Fc domain via a short linker. Figure 2 shows activation of Jurkat / NFAT-Luc / Cl 3C7 / HPV16E7(11-19)TCR709 cells by IgG1-containing T cell activator constructs. [Figure 15B]Figure 1 shows activation of Jurkat reporter cells by T cell activator constructs with IgG1 or IgG4 Fc domains, where each pMHC complex is connected to the hinge region of the IgG1 or IgG4 Fc domain via a short linker. Figure 2 shows activation of Jurkat / NFAT-Luc / Cl 3C7 / HPV16E6(29-38) TCR cells by IgG1-containing T cell activator constructs. [Figure 15C] Figure 1 shows activation of Jurkat reporter cells by T cell activator constructs with IgG1 or IgG4 Fc domains, where each pMHC complex is connected to the hinge region of the IgG1 or IgG4 Fc domain via a short linker. Figure 2 shows activation of Jurkat / NFAT-Luc / Cl 3C7 / HPV16E7(11-19)TCR709 cells by IgG4-containing T cell activator constructs. [Figure 15D] Figure 1 shows activation of Jurkat reporter cells by T cell activator constructs with IgG1 or IgG4 Fc domains, where each pMHC complex is connected to the hinge region of the IgG1 or IgG4 Fc domain via a short linker. Figure 2 shows activation of Jurkat / NFAT-Luc / Cl 3C7 / HPV16E6(29-38) TCR cells by IgG4-containing T cell activator constructs. [Figure 16A] Figure 1 shows activation of Jurkat reporter cells by T cell activator constructs with IgG1 or IgG4 Fc domains, where each pMHC complex is connected to the hinge region of the IgG1 or IgG4 Fc domain via a long linker. Figure 2 shows activation of Jurkat / NFAT-Luc / Cl 3C7 / HPV16E7(11-19)TCR709 cells by IgG1-containing T cell activator constructs. [Figure 16B] Figure 1 shows activation of Jurkat reporter cells by T cell activator constructs with IgG1 or IgG4 Fc domains, where each pMHC complex is connected to the hinge region of the IgG1 or IgG4 Fc domain via a long linker. Figure 2 shows activation of Jurkat / NFAT-Luc / Cl 3C7 / HPV16E6(29-38) TCR cells by IgG1-containing T cell activator constructs. [Figure 16C] Figure 1 shows activation of Jurkat reporter cells by T cell activator constructs with IgG1 or IgG4 Fc domains, where each pMHC complex is connected to the hinge region of the IgG1 or IgG4 Fc domain via a long linker. Figure 2 shows activation of Jurkat / NFAT-Luc / Cl 3C7 / HPV16E7(11-19)TCR709 cells by IgG4-containing T cell activator constructs. [Figure 16D] Figure 1 shows activation of Jurkat reporter cells by T cell activator constructs with IgG1 or IgG4 Fc domains, where each pMHC complex is connected to the hinge region of the IgG1 or IgG4 Fc domain via a long linker. Figure 2 shows activation of Jurkat / NFAT-Luc / Cl 3C7 / HPV16E6(29-38) TCR cells by IgG4-containing T cell activator constructs. [Figure 17A] Figure 1 shows activation of Jurkat reporter cells by T cell activator constructs, each containing a pMHC complex and the antigen-binding domain of an anti-CD3 antibody. Figure 2 shows activation of Jurkat / NFAT-Luc / Cl 3C7 / HPV16E7(11-19)TCR709 cells by T cell activator constructs, each containing a pMHC complex connected to the hinge region of the Fc domain via a short linker. [Figure 17B] Figure 1 shows activation of Jurkat reporter cells by T cell activator constructs, each containing a pMHC complex and the antigen-binding domain of an anti-CD3 antibody. Figure 2 shows activation of Jurkat / NFAT-Luc / Cl 3C7 / HPV16E6(29-38) TCR cells by T cell activator constructs, each containing a pMHC complex connected to the hinge region of the Fc domain via a short linker. [Figure 17C] Figure 1 shows activation of Jurkat reporter cells by T cell activator constructs, each containing a pMHC complex and the antigen-binding domain of an anti-CD3 antibody. Figure 2 shows activation of Jurkat / NFAT-Luc / Cl 3C7 / HPV16E7(11-19)TCR709 cells by T cell activator constructs, each containing a pMHC complex connected to the hinge region of the Fc domain via a long linker. [Figure 17D] Figure 1 shows activation of Jurkat reporter cells by T cell activator constructs, each containing a pMHC complex and the antigen-binding domain of an anti-CD3 antibody. Figure 2 shows activation of Jurkat / NFAT-Luc / Cl 3C7 / HPV16E6(29-38) TCR cells by T cell activator constructs, each containing a pMHC complex connected to the hinge region of the Fc domain via a long linker. [Figure 18A] Figure 1 shows histograms showing antigen-specific expansion of CMVpp65 T cells upon treatment with low concentrations of T cell activators containing long linkers. CMVpp65 tetramer+ T cells are represented by the light-shaded curve, CMVpp65 tetramer- CD8+ T cells are represented by the dark-shaded curve, and non-expanded CD8+ CMV PBMCs (sham control) are represented by the dotted curve. CMVpp65-peptide-pulsed cell populations are shown. [Figure 18B] Figure 1 shows histograms showing antigen-specific expansion of CMVpp65 T cells upon treatment with low concentrations of T cell activators containing long linkers. CMVpp65 tetramer+ T cells are represented by the light-shaded curve, CMVpp65 tetramer-CD8+ T cells are represented by the dark-shaded curve, and non-expanded CD8+ CMV PBMCs (sham control) are represented by the dotted curve. Cell populations treated with 0.02 nM CMVpp65 pMHCxCD20 T cell activators containing long linkers are shown. [Figure 18C] Figure 1 shows histograms showing antigen-specific expansion of CMVpp65 T cells upon treatment with low concentrations of T cell activators containing long linkers. CMVpp65 tetramer+ T cells are represented by the light-shaded curve, CMVpp65 tetramer-CD8+ T cells are represented by the dark-shaded curve, and non-expanded CD8+ CMV PBMCs (sham control) are represented by the dotted curve. Cell populations treated with 1.8 nM CMVpp65 pMHCxCD20 T cell activators containing long linkers are shown. [Figure 18D]This histogram shows the antigen-specific expansion of CMVpp65 T cells upon treatment with low concentrations of T cell activators containing long linkers. CMVpp65 tetramer+ T cells are represented by the light-shaded curve, CMVpp65 tetramer-CD8+ T cells are represented by the dark-shaded curve, and non-expanded CD8+ CMV PBMCs (sham control) are represented by the dotted curve. Cell populations treated with 0.2 nM MAGE-A4 pMHCxCD20 containing long linkers are shown. [Figure 18E] This histogram shows the antigen-specific expansion of CMVpp65 T cells upon treatment with low concentrations of T cell activators containing long linkers. CMVpp65 tetramer+ T cells are represented by the light-shaded curve, CMVpp65 tetramer-CD8+ T cells are represented by the dark-shaded curve, and non-expanded CD8+ CMV PBMCs (sham control) are represented by the dotted curve. Cell populations treated with 1.8 nM MAGE-A4 pMHCxCD20 containing long linkers are shown. [Figure 18F] Figure 1 shows histograms showing antigen-specific expansion of CMVpp65 T cells upon treatment with low concentrations of T cell activators containing long linkers. CMVpp65 tetramer+ T cells are represented by the light-shaded curve, CMVpp65 tetramer- CD8+ T cells are represented by the dark-shaded curve, and non-expanded CD8+ CMV PBMCs (sham control) are represented by the dotted curve. The percentage of proliferation in tetramer+ and tetramer- cells upon treatment with CMVpp65 pMHCxCD20 T cell activators containing long linkers is shown. [Figure 18G] Figure 1 shows histograms depicting antigen-specific expansion of CMVpp65 T cells upon treatment with low concentrations of T cell activators containing long linkers. CMVpp65 tetramer+ T cells are represented by the light-shaded curve, CMVpp65 tetramer-CD8+ T cells are represented by the dark-shaded curve, and non-expanded CD8+ CMV PBMCs (sham control) are represented by the dotted curve. Figure 2 shows B cell killing upon treatment with various concentrations of CMVpp65 pMHCxCD20 T cell activators containing long linkers. [Figure 19A]Figure 1 shows histograms showing antigen-specific expansion of CMVpp65 T cells upon treatment with low concentrations of T cell activators containing short linkers. CMVpp65 tetramer+ T cells are represented by the light-shaded curve, CMVpp65 tetramer- CD8+ T cells are represented by the dark-shaded curve, and non-expanded CD8+ CMV PBMCs (sham control) are represented by the dotted curve. CMVpp65 peptide-pulsed cell populations are shown. [Figure 19B] Figure 1 shows histograms showing antigen-specific expansion of CMVpp65 T cells upon treatment with low concentrations of T cell activators containing short linkers. CMVpp65 tetramer+ T cells are represented by the light-shaded curve, CMVpp65 tetramer-CD8+ T cells are represented by the dark-shaded curve, and non-expanded CD8+ CMV PBMCs (sham control) are represented by the dotted curve. Cell populations treated with 0.02 nM CMVpp65 pMHCxCD20 T cell activators containing short linkers are shown. [Figure 19C] Figure 1 shows histograms showing antigen-specific expansion of CMVpp65 T cells upon treatment with low concentrations of T cell activators containing short linkers. CMVpp65 tetramer+ T cells are represented by the light-shaded curve, CMVpp65 tetramer- CD8+ T cells are represented by the dark-shaded curve, and non-expanded CD8+ CMV PBMCs (sham control) are represented by the dotted curve. Cell populations treated with 1.8 nM CMVpp65 pMHCxCD20 T cell activators containing short linkers are shown. [Figure 19D] This is a histogram showing antigen-specific expansion of CMVpp65 T cells upon treatment with low concentrations of T cell activators containing short linkers. CMVpp65 tetramer+ T cells are represented by the light-shaded curve, CMVpp65 tetramer-CD8+ T cells are represented by the dark-shaded curve, and non-expanded CD8+ CMV PBMCs (sham control) are represented by the dotted curve. Cell populations treated with 0.2 nM MAGE-A4 pMHCxCD20 containing short linkers are shown. [Figure 19E]This histogram shows antigen-specific expansion of CMVpp65 T cells upon treatment with low concentrations of T cell activators containing short linkers. CMVpp65 tetramer+ T cells are represented by the light-shaded curve, CMVpp65 tetramer-CD8+ T cells are represented by the dark-shaded curve, and non-expanded CD8+ CMV PBMCs (sham control) are represented by the dotted curve. Cell populations treated with 1.8 nM MAGE-A4 pMHCxCD20 containing short linkers are shown. [Figure 19F] Figure 1 shows histograms showing antigen-specific expansion of CMVpp65 T cells upon treatment with low concentrations of T cell activators containing short linkers. CMVpp65 tetramer+ T cells are represented by the light-shaded curve, CMVpp65 tetramer- CD8+ T cells are represented by the dark-shaded curve, and non-expanded CD8+ CMV PBMCs (sham control) are represented by the dotted curve. The percentage of proliferation in tetramer+ and tetramer- cells upon treatment with CMVpp65 pMHCxCD20 T cell activators containing short linkers is shown. [Figure 19G] Figure 1 shows histograms depicting antigen-specific expansion of CMVpp65 T cells upon treatment with low concentrations of T cell activators containing short linkers. CMVpp65 tetramer+ T cells are represented by the light-shaded curve, CMVpp65 tetramer-CD8+ T cells are represented by the dark-shaded curve, and non-expanded CD8+ CMV PBMCs (sham control) are represented by the dotted curve. Figure 2 shows B cell killing upon treatment with various concentrations of CMVpp65 pMHCxCD20 T cell activators containing short linkers. [Figure 20A] Figure 1 shows antigen-specific expansion of CMVpp65 T cells upon treatment with low concentrations of T cell activators containing CMVpp65 pMHC, as determined by flow cytometry, as well as the frequency of CD8+ tetramer+ cells in CMVpp65 cells treated with 0.02 nM CMVpp65 pMHC x CD20 T cell activators containing a long linker. [Figure 20B]Figure 1 shows antigen-specific expansion of CMVpp65 T cells upon treatment with low concentrations of T cell activators containing CMVpp65 pMHC, as determined by flow cytometry, as well as the frequency of CD8+ tetramer+ cells in CMVpp65 cells treated with 1.8 nM CMVpp65 pMHC x CD20 T cell activators containing a long linker. [Figure 20C] Figure 1 shows antigen-specific expansion of CMVpp65 T cells upon treatment with low concentrations of T cell activators containing CMVpp65 pMHC, as determined by flow cytometry. Figure 2 shows the frequency of CD8+ tetramer+ cells in CMVpp65 peptide+IFNα-pulsed CMVpp65+ T cells. [Figure 20D] Figure 1 shows antigen-specific expansion of CMVpp65 T cells upon treatment with low concentrations of T cell activators containing CMVpp65 pMHC, as determined by flow cytometry, and shows the frequency of CD8+ tetramer+ cells in CMVpp65 cells treated with 0.02 nM MAGE-A4 pMHC x CD20 control T cell activator containing a long linker. [Figure 20E] Figure 1 shows antigen-specific expansion of CMVpp65 T cells upon treatment with low concentrations of T cell activators containing CMVpp65 pMHC, as determined by flow cytometry, and shows the frequency of CD8+ tetramer+ cells in CMVpp65 cells treated with 1.8 nM MAGE-A4 pMHC x CD20 control T cell activators containing a long linker. [Figure 20F] Figure 1 shows antigen-specific expansion of CMVpp65 T cells upon treatment with low concentrations of T cell activators containing CMVpp65 pMHC, as determined by flow cytometry, as well as the frequency of CD8+ tetramer+ cells in CMVpp65 cells treated with 0.02 nM CMVpp65 pMHC x CD20 T cell activators containing a short linker. [Figure 20G]Figure 1 shows antigen-specific expansion of CMVpp65 T cells upon treatment with low concentrations of T cell activators containing CMVpp65 pMHC, as determined by flow cytometry. Figure 2 shows the frequency of CD8+ tetramer+ cells in CMVpp65 cells treated with 1.8 nM CMVpp65 pMHC x CD20 T cell activators containing a short linker. [Figure 20H] Figure 1 shows antigen-specific expansion of CMVpp65 T cells upon treatment with low concentrations of T cell activators containing CMVpp65 pMHC, shown as the frequency of T cells as determined by flow cytometry. Figure 2 shows the frequency of CD8+ tetramer+ cells in non-expanded CMV PBMCs. [Figure 20I] Figure 1 shows antigen-specific expansion of CMVpp65 T cells upon treatment with low concentrations of T cell activators containing CMVpp65 pMHC, as determined by flow cytometry, and shows the frequency of CD8+ tetramer+ cells in CMVpp65 cells treated with 0.02 nM MAGE-A4 pMHC x CD20 control T cell activator containing a short linker. [Figure 20J] Figure 1 shows antigen-specific expansion of CMVpp65 T cells upon treatment with low concentrations of T cell activators containing CMVpp65 pMHC, as determined by flow cytometry, and shows the frequency of CD8+ tetramer+ cells in CMVpp65 cells treated with 1.8 nM MAGE-A4 pMHC x CD20 control T cell activator containing a short linker. [Figure 21A] Figures 21A-21E show histograms demonstrating antigen-specific expansion of CMVpp65 T cells upon treatment with low concentrations of T cell activators in non-depleted PBMCs (Figures 21A-21E), PBMCs with B cell depletion (Figures 21F-21H), and isolated Pan T cells (Figures 21I-21K). CMVpp65 tetramer+ T cells are represented by the light-shaded curve, CMVpp65 tetramer- CD8+ T cells are represented by the dark-shaded curve, and non-expanded CD8+ CMV PBMCs (sham control) are represented by the dotted curve. CMVpp65 peptide-pulsed cell populations are shown. [Figure 21B]Figures 21A-21E show histograms demonstrating antigen-specific expansion of CMVpp65 T cells upon treatment with low concentrations of T cell activators in non-depleted PBMCs (Figures 21A-21E), PBMCs with B cell depletion (Figures 21F-21H), and isolated Pan T cells (Figures 21I-21K). CMVpp65 tetramer T cells are represented by the light-shaded curve, CMVpp65 tetramer CD8 T cells are represented by the dark-shaded curve, and non-expanded CD8 CMV PBMCs (sham control) are represented by the dotted curve. Cell populations with a 1:5 CMV T cell:B cell ratio treated with 0.02 nM CMVpp65 pMHCxCD20 T cell activator containing a long linker are shown. [Figure 21C] Figures 21A-21E show histograms demonstrating antigen-specific expansion of CMVpp65 T cells upon treatment with low concentrations of T cell activators in undepleted PBMCs (Figures 21A-21E), PBMCs with B cell depletion (Figures 21F-21H), and isolated Pan T cells (Figures 21I-21K). CMVpp65 tetramer+ T cells are represented by the light-shaded curve, CMVpp65 tetramer-CD8+ T cells are represented by the dark-shaded curve, and non-expanded CD8+ CMV PBMCs (sham control) are represented by the dotted curve. Cell populations with a 1:5 CMV T cell:B cell ratio treated with 0.02 nM CMVpp65 pMHCxCD20 T cell activator containing a short linker are shown. [Figure 21D] Figures 21A-21E show histograms demonstrating antigen-specific expansion of CMVpp65 T cells upon treatment with low concentrations of T cell activators in non-depleted PBMCs (Figures 21A-21E), PBMCs with B cell depletion (Figures 21F-21H), and isolated Pan T cells (Figures 21I-21K). CMVpp65 tetramer T cells are represented by the light-shaded curve, CMVpp65 tetramer CD8 T cells are represented by the dark-shaded curve, and non-expanded CD8 CMV PBMCs (sham control) are represented by the dotted curve. Cell populations with a 1:5 CMV T cell:B cell ratio treated with 0.02 nM MAGE-A4 pMHCxCD20 T cell activator containing a long linker are shown. [Figure 21E]Figures 21A-21E show histograms demonstrating antigen-specific expansion of CMVpp65 T cells upon treatment with low concentrations of T cell activators in non-depleted PBMCs (Figures 21A-21E), PBMCs with B cell depletion (Figures 21F-21H), and isolated Pan T cells (Figures 21I-21K). CMVpp65 tetramer+ T cells are represented by the light-shaded curve, CMVpp65 tetramer- CD8+ T cells are represented by the dark-shaded curve, and non-expanded CD8+ CMV PBMCs (sham control) are represented by the dotted curve. Cell populations with a 1:5 CMV T cell:B cell ratio treated with 0.02 nM MAGE-A4 pMHCxCD20 T cell activator containing a short linker are shown. [Figure 21F] Figures 21A-21E show histograms demonstrating antigen-specific expansion of CMVpp65 T cells upon treatment with low concentrations of T cell activators in non-depleted PBMCs (Figures 21A-21E), PBMCs with B cell depletion (Figures 21F-21H), and isolated Pan T cells (Figures 21I-21K). CMVpp65 tetramer+ T cells are represented by the light-shaded curve, CMVpp65 tetramer- CD8+ T cells are represented by the dark-shaded curve, and non-expanded CD8+ CMV PBMCs (sham control) are represented by the dotted curve. CMVpp65 peptide-pulsed cell populations are shown. [Figure 21G] Figures 21A-21E show histograms demonstrating antigen-specific expansion of CMVpp65 T cells upon treatment with low concentrations of T cell activators in non-depleted PBMCs (Figures 21A-21E), PBMCs with B cell depletion (Figures 21F-21H), and isolated Pan T cells (Figures 21I-21K). CMVpp65 tetramer T cells are represented by the light-shaded curve, CMVpp65 tetramer CD8 T cells are represented by the dark-shaded curve, and non-expanded CD8 CMV PBMCs (sham control) are represented by the dotted curve. Cell populations with a 1:5 CMV T cell:B cell ratio treated with 0.02 nM CMVpp65 pMHCxCD20 T cell activator containing a long linker are shown. [Figure 21H]Figures 21A-21E show histograms demonstrating antigen-specific expansion of CMVpp65 T cells upon treatment with low concentrations of T cell activators in undepleted PBMCs (Figures 21A-21E), PBMCs with B cell depletion (Figures 21F-21H), and isolated Pan T cells (Figures 21I-21K). CMVpp65 tetramer+ T cells are represented by the light-shaded curve, CMVpp65 tetramer-CD8+ T cells are represented by the dark-shaded curve, and non-expanded CD8+ CMV PBMCs (sham control) are represented by the dotted curve. Cell populations with a 1:5 CMV T cell:B cell ratio treated with 0.02 nM CMVpp65 pMHCxCD20 T cell activator containing a short linker are shown. [Figure 21I] Figures 21A-21E show histograms demonstrating antigen-specific expansion of CMVpp65 T cells upon treatment with low concentrations of T cell activators in non-depleted PBMCs (Figures 21A-21E), PBMCs with B cell depletion (Figures 21F-21H), and isolated Pan T cells (Figures 21I-21K). CMVpp65 tetramer+ T cells are represented by the light-shaded curve, CMVpp65 tetramer- CD8+ T cells are represented by the dark-shaded curve, and non-expanded CD8+ CMV PBMCs (sham control) are represented by the dotted curve. CMVpp65 peptide-pulsed cell populations are shown. [Figure 21J] Figures 21A-21E show histograms demonstrating antigen-specific expansion of CMVpp65 T cells upon treatment with low concentrations of T cell activators in non-depleted PBMCs (Figures 21A-21E), PBMCs with B cell depletion (Figures 21F-21H), and isolated Pan T cells (Figures 21I-21K). CMVpp65 tetramer+ T cells are represented by the light-shaded curve, CMVpp65 tetramer-CD8+ T cells are represented by the dark-shaded curve, and non-expanded CD8+ CMV PBMCs (sham control) are represented by the dotted curve. Cell populations treated with 0.02 nM CMVpp65 pMHCxCD20 T cell activator containing a long linker are shown. [Figure 21K]Figures 21A-21E show histograms demonstrating antigen-specific expansion of CMVpp65 T cells upon treatment with low concentrations of T cell activators in non-depleted PBMCs (Figures 21A-21E), PBMCs with B cell depletion (Figures 21F-21H), and isolated Pan T cells (Figures 21I-21K). CMVpp65 tetramer+ T cells are represented by the light-shaded curve, CMVpp65 tetramer- CD8+ T cells are represented by the dark-shaded curve, and non-expanded CD8+ CMV PBMCs (sham control) are represented by the dotted curve. Cell populations treated with 0.02 nM CMVpp65 pMHCxCD20 T cell activator containing a short linker are shown. [Figure 22A] Figure 1 shows histograms showing antigen-specific expansion of CMVpp65 T cells upon treatment of PBMCs with a low concentration of T cell activator at a CMV T cell:B cell ratio of 1:58. CMVpp65 tetramer+ T cells are represented by the light-shaded curve, CMVpp65 tetramer- CD8+ T cells are represented by the dark-shaded curve, and non-expanded CD8+ CMV PBMCs (sham control) are represented by the dotted curve. CMVpp65 peptide-pulsed cell populations are shown. [Figure 22B] Histograms show antigen-specific expansion of CMVpp65 T cells upon treatment of PBMCs at a CMV T cell:B cell ratio of 1:58 with low concentrations of T cell activators. CMVpp65 tetramer+ T cells are represented by the light-shaded curve, CMVpp65 tetramer-CD8+ T cells are represented by the dark-shaded curve, and non-expanded CD8+ CMV PBMCs (sham control) are represented by the dotted curve. Cell populations treated with 0.02 nM CMVpp65 pMHCxCD20 T cell activator containing a long linker are shown. [Figure 22C] Histograms show antigen-specific expansion of CMVpp65 T cells upon treatment of PBMCs at a CMV T cell:B cell ratio of 1:58 with low concentrations of T cell activators. CMVpp65 tetramer+ T cells are represented by the light-shaded curve, CMVpp65 tetramer-CD8+ T cells are represented by the dark-shaded curve, and non-expanded CD8+ CMV PBMCs (sham control) are represented by the dotted curve. Cell populations treated with 0.02 nM CMVpp65 pMHCxCD20 T cell activator containing a short linker are shown. [Figure 22D] Histogram showing antigen-specific expansion of CMVpp65 T cells upon treatment of PBMCs at a CMV T cell:B cell ratio of 1:58 with low concentrations of T cell activators. CMVpp65 tetramer+ T cells are represented by the light-shaded curve, CMVpp65 tetramer-CD8+ T cells are represented by the dark-shaded curve, and non-expanded CD8+ CMV PBMCs (sham control) are represented by the dotted curve. Cell populations treated with 0.02 nM MAGE-A4 pMHC×CD20 T cell activator containing a long linker are shown. [Figure 22E] Histograms show antigen-specific expansion of CMVpp65 T cells upon treatment of PBMCs with a low concentration of T cell activator at a CMV T cell:B cell ratio of 1:58. CMVpp65 tetramer+ T cells are represented by the light-shaded curve, CMVpp65 tetramer-CD8+ T cells are represented by the dark-shaded curve, and non-expanded CD8+ CMV PBMCs (sham control) are represented by the dotted curve. Cell populations treated with 0.02 nM MAGE-A4 pMHC×CD20 T cell activator containing a short linker are shown. [Figure 22F] Histograms showing antigen-specific expansion of CMVpp65 T cells upon treatment of PBMCs at a CMV T cell:B cell ratio of 1:58 with low concentrations of T cell activators. CMVpp65 tetramer+ T cells are represented by the light-shaded curve, CMVpp65 tetramer-CD8+ T cells are represented by the dark-shaded curve, and non-expanded CD8+ CMV PBMCs (sham control) are represented by the dotted curve. B cell killing upon treatment with various concentrations of CMVpp65 pMHCxCD20 T cell activators is shown. LL: long linker, SL: short linker. [Figure 22G]Figure 1 shows histograms showing antigen-specific expansion of CMVpp65 T cells upon treatment of PBMCs with a low concentration of T cell activator at a CMV T cell:B cell ratio of 1:58. CMVpp65 tetramer+ T cells are represented by the light-shaded curve, CMVpp65 tetramer-CD8+ T cells are represented by the dark-shaded curve, and non-expanded CD8+ CMV PBMCs (sham control) are represented by the dotted curve. B cell killing is shown upon treatment with parental anti-CD3xanti-CD20 antibody (also referred to herein as CD3xCD20 or REGN1979) containing either a short or long linker or CMVpp65 pMHCxCD20 T cell activator. LL: long linker; SL: short linker. [Figure 22H] Histogram showing antigen-specific expansion of CMVpp65 T cells upon treatment of PBMCs at a CMV T cell:B cell ratio of 1:58 with low concentrations of T cell activators. CMVpp65 tetramer+ T cells are represented by the light-shaded curve, CMVpp65 tetramer- CD8+ T cells are represented by the dark-shaded curve, and non-expanded CD8+ CMV PBMCs (sham control) are represented by the dotted curve. Cell populations treated with the parental antibody REGN1979 are shown. [Figure 22I] Figure 1 shows histograms showing antigen-specific expansion of CMVpp65 T cells upon treatment of PBMCs with a low concentration of T cell activator at a CMV T cell:B cell ratio of 1:58. CMVpp65 tetramer+ T cells are represented by the light-shaded curve, CMVpp65 tetramer- CD8+ T cells are represented by the dark-shaded curve, and non-expanded CD8+ CMV PBMCs (sham control) are represented by the dotted curve. CMVpp65 peptide-pulsed cell populations are shown. [Figure 23A] 1 is a histogram showing antigen-specific expansion of CMVpp65 T cells upon treatment with low concentrations of T cell activators bearing different immune cell antigen-targeting moieties. CMVpp65 peptide-pulsed cell populations are shown. [Figure 23B]1 is a histogram showing antigen-specific expansion of CMVpp65 T cells upon treatment with low concentrations of T cell activators with different immune cell antigen-targeting moieties. Shown is a cell population treated with 0.02 nM CMVpp65 pMHC×CD20 T cell activator containing a long linker. [Figure 23C] 1 is a histogram showing antigen-specific expansion of CMVpp65 T cells upon treatment with low concentrations of T cell activators with different immune cell antigen-targeting moieties. Shown is a cell population treated with 0.02 nM CMVpp65 pMHC×CD20 T cell activator containing a short linker. [Figure 23D] 1 is a histogram showing antigen-specific expansion of CMVpp65 T cells upon treatment with low concentrations of T cell activators with different immune cell antigen-targeting moieties. Shown is a cell population treated with 0.02 nM MAGE-A4 pMHC×CD20 T cell activator containing a long linker. [Figure 23E] 1 is a histogram showing antigen-specific expansion of CMVpp65 T cells upon treatment with low concentrations of T cell activators with different immune cell antigen-targeting moieties. Shown is a cell population treated with 0.02 nM MAGE-A4 pMHC×CD20 T cell activator containing a short linker. [Figure 23F] 1 is a histogram showing antigen-specific expansion of CMVpp65 T cells upon treatment with low concentrations of T cell activators with different immune cell antigen-targeting moieties. Cell populations treated with 0.2 nM CMVpp65 pMHC×CD3 T cell activators containing long linkers are shown. [Figure 23G] 1 is a histogram showing antigen-specific expansion of CMVpp65 T cells upon treatment with low concentrations of T cell activators with different immune cell antigen-targeting moieties. Cell populations treated with 0.2 nM CMVpp65 pMHC×CD3 T cell activators containing short linkers are shown. [Figure 23H]1 is a histogram showing antigen-specific expansion of CMVpp65 T cells upon treatment with low concentrations of T cell activators with different immune cell antigen-targeting moieties. Cell populations treated with 0.2 nM MAGE-A4 pMHC×CD3 T cell activators containing long linkers are shown. [Figure 23I] 1 is a histogram showing antigen-specific expansion of CMVpp65 T cells upon treatment with low concentrations of T cell activators with different immune cell antigen-targeting moieties. Cell populations treated with 0.2 nM MAGE-A4 pMHC×CD3 T cell activators containing short linkers are shown. DETAILED DESCRIPTION OF THE INVENTION

[0015] 6.1.Definition About, Approximately: The terms "about," "approximately," and the like are used throughout the specification before a numerical value to indicate that the numerical value is not necessarily exact (e.g., to account for fractions, variations in measurement precision and / or accuracy, timing, etc.). A disclosure of "about X" or "approximately X," where X is a numerical value, should be understood to also be a disclosure of "X." Thus, for example, disclosure of embodiments in which a sequence has "about X% sequence identity" to another sequence is also a disclosure of embodiments in which the sequence has "X% sequence identity" to the other sequence.

[0016] And / Or: Unless otherwise indicated, the conjunction "or" is intended to be used in its proper sense as a Boolean logic operator, encompassing both the selection of features in an alternative (selection of A is mutually exclusive from B, A OR B) and the selection of conjunctive features (selection of both A and B, A OR B). In several places in the text, the term "and / or" is used interchangeably and should not be construed to mean that "or" is used in reference to mutually exclusive alternatives.

[0017] Antigen-binding domain or ABD: As used herein, the term "antigen-binding domain" or "ABD" refers to the portion of a targeting moiety that can specifically, non-covalently, and reversibly bind to a target molecule.

[0018] Antibody: As used herein, the term "antibody" refers to a polypeptide (or set of polypeptides) of the immunoglobulin family that can non-covalently, reversibly, and specifically bind to an antigen. For example, a naturally occurring "antibody" of the IgG type is a tetramer containing at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is composed of three domains: CH1, CH2, and CH3. Each light chain is composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is composed of one domain (abbreviated herein as CL). The VH and VL regions can be further subdivided into regions of hypervariability called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant regions of the antibody may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. The term "antibody" includes, but is not limited to, monoclonal antibodies, human antibodies, humanized antibodies, camelized antibodies, chimeric antibodies, bispecific or multispecific antibodies, and anti-idiotypic (anti-id) antibodies. Antibodies can be of any isotype / class (e.g., IgG, IgE, IgM, IgD, IgA, and IgY) or subclass (e.g., lgG1, lgG2, lgG3, lgG4, lgA1, and lgA2). Both the light and heavy chains are divided into regions of structural and functional homology. The terms "constant" and "variable" are used functionally. In this regard, it will be understood that the variable domains of both the light (VL) and heavy (VH) chain portions determine antigen recognition and specificity.Conversely, the constant domains of the light chain (CL) and heavy chain (CH1, CH2, or CH3) confer important biological properties, such as secretion, placental transport, Fc receptor binding, and complement fixation. By convention, the numbering of constant region domains increases as they become more distal from the antigen-binding site or amino-terminus of the antibody. The N-terminus is the variable region, the C-terminus is the constant region, and the CH3 and CL domains represent the carboxy-termini of the heavy and light chains of native antibodies, respectively. For convenience, and unless otherwise indicated by context, reference to an antibody also refers to antibody fragments and engineered antibodies, including molecules having non-naturally occurring antigen-binding sites and / or antigen-binding sites with non-native configurations, e.g., Fab or scFv domains C-terminal to the CH3 domain.

[0019] Antigenic peptide: As used herein, the term "antigenic peptide" (also used interchangeably as "antigenic peptide" or "antigenic determinant") relates to a portion or fragment of an antigen that can stimulate an immune response, preferably a cellular response, against the antigen or against cells characterized by expression of the antigen (e.g., cancer cells). In some embodiments, the antigenic peptide is capable of stimulating a cellular response against cells characterized by expression or presentation of the antigen, preferably capable of activating antigen-responsive cytotoxic T lymphocytes (CTLs). In certain embodiments, the antigenic peptide is a peptide from LCMV, such as gp33-41, APF(126-134), BALF(276-284), CEA(571-579), CMV pp65(495-503), FLU-M1(58-66), gp100(154-162), gp100(209-217), HBV Core(18-27), Her2 / neu(369-377; V2v9); HPV E7(11-20), HPV E7(11-19), HPV and portions or fragments of tumor antigens, including, but not limited to, E7(82-90), KLK4(11-19), LMP1(125-133), MAG-A3(112-120), MAGE-A4(230-239), MAGE-A4(286-294), NYESO1(157-165, C165A), NYESO1(157-165, C165V), p54 WT(264-272), PAP-3(136-143), PSMA(4-12), PSMA(135-145), survivin(96-014), tyrosinase(369-377, 371D), and WT1(126-134). In some embodiments, the antigenic peptide is 7 to 20 amino acids in length, 7 to 12 amino acids in length, 8 to 11 amino acids in length, or 9 to 10 amino acids in length. In some embodiments, the antigenic peptide is 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length.

[0020] Associated: The term "associated" in the context of a T cell activator or component thereof refers to a functional relationship between two or more polypeptide chains. Specifically, the term "associated" means that two or more polypeptides are associated with one another, e.g., non-covalently via molecular interactions or covalently via one or more disulfide or chemical crosslinks, to generate a functional T cell activator. Examples of associations that may be present in the T cell activators of the present disclosure include, but are not limited to, associations between homodimeric or heterodimeric Fc domains within an Fc region (homodimers or heterodimers are as described in Section 6.7.1), associations between VH and VL regions within a Fab or scFv, associations between CH1 and CL within a Fab, and associations between CH3 and CH3 within a domain-substituted Fab.

[0021] B cell antigen: As used herein, the term "B cell antigen" refers to any biological molecule (e.g., a protein, carbohydrate, lipid, or a combination thereof) present on and / or expressed by a B cell. In some embodiments, at least a portion of the B cell antigen is extracellular (e.g., a cell surface protein or a transmembrane protein having at least one extracellular domain). Specific B cell antigens contemplated herein include, but are not limited to, CD19, CD20, and CD22.

[0022] Bivalent: As used herein with respect to a pMHC complex and / or targeting moiety in a T cell activator, the term "bivalent" refers to a T cell activator having two pMHC complexes and / or targeting moieties, respectively. In some embodiments, a bivalent T cell activator with respect to a pMHC complex and / or targeting moiety is a dimer (either a homodimer or a heterodimer).

[0023] Cancer: The term "cancer" refers to a disease characterized by the uncontrolled (and often rapid) growth of abnormal cells. Cancer cells can spread locally or to other parts of the body via the bloodstream and lymphatic system. Examples of various cancers are described herein, including, but not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, brain cancer, adrenal gland cancer, autonomic ganglion cancer, biliary tract cancer, bone cancer, endometrial cancer, eye cancer, fallopian tube cancer, reproductive tract cancer, colon cancer, meningeal cancer, esophageal cancer, peritoneal cancer, pituitary cancer, penile cancer, placental cancer, pleural cancer, salivary gland cancer, small intestine cancer, stomach cancer, testicular cancer, thymus cancer, thyroid cancer, upper aerodigestive tract cancer, urinary tract cancer, vaginal cancer, vulvar cancer, lymphoma, leukemia, lung cancer, and the like.

[0024] Complementarity determining region or CDR: As used herein, the term "complementarity determining region" or "CDR" refers to the amino acid sequence in an antibody variable region that confers antigen specificity and binding affinity. For example, typically, each heavy chain variable region has three CDRs (CDR-H1, CDR-H2, CDR-H3), and each light chain variable region has three CDRs (CDR1-L1, CDR-L2, CDR-L3). The precise amino acid sequence boundaries of a given CDR can be determined according to Kabat et al., 1991, "Sequences of Proteins of Immunological Interest," 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD ("Kabat" numbering scheme), Al-Lazikani et al., 1997, JMB 273:927-948 ("Chothia" numbering scheme), and ImMunoGeneTics (IMGT) numbering (Lefranc, 1999, The Immunologist 7:132-136; Lefranc et al. The CDR numbering scheme may be determined using any of several well-known schemes, including those described by Kabat et al., 2003, Dev. Comp. Immunol. 27:55-77 ("IMGT" numbering scheme). For example, under Kabat, for the classical format, the CDR amino acid residues within the heavy chain variable domain (VH) are numbered 31-35 (CDR-H1), 50-65 (CDR-H2), and 95-102 (CDR-H3), and the CDR amino acid residues within the light chain variable domain (VH) are numbered 31-35 (CDR-H1), 50-65 (CDR-H2), and 95-102 (CDR-H3). The CDR amino acid residues within the VH are numbered 24-34 (CDR-L1), 50-56 (CDR-L2), and 89-97 (CDR-L3). Under Chothia, the CDR amino acids within the VH are numbered 26-32 (CDR-H1), 52-56 (CDR-H2), and 95-102 (CDR-H3), and the amino acid residues within the VL are numbered 26-32 (CDR-L1), 50-52 (CDR-L2), and 91-96 (CDR-L3).Combining the CDR definitions of both Kabat and Chothia, the CDRs consist of amino acid residues 26-35 (CDR-H1), 50-65 (CDR-H2), and 95-102 (CDR-H3) in human VH, and amino acid residues 24-34 (CDR-L1), 50-56 (CDR-L2), and 89-97 (CDR-L3) in human VL. Under IMGT, the CDR amino acid residues in VH are numbered approximately 26-35 (CDR-H1), 51-57 (CDR-H2), and 93-102 (CDR-H3), and the CDR amino acid residues in VL are numbered approximately 27-32 (CDR-L1), 50-52 (CDR-L2), and 89-97 (CDR-L3) ("Kabat" numbering). Under IMGT, the CDR regions of an antibody can be determined using the program IMGT / DomainGap Align.

[0025] EC50: The term "EC50" refers to the half maximal effective concentration of a molecule (such as an antibody or T cell activator) that induces a response halfway between baseline and maximum after a specified exposure time. EC50 essentially represents the concentration of a molecule (e.g., an antibody or T cell activator) at which 50% of its maximal effect is observed. In certain embodiments, the EC50 value is equal to the concentration of a T cell activator that gives half-maximal T cell activation in the assay described in Section 8.1.2.

[0026] Epitope: An epitope, or antigenic determinant, is the portion of an antigen (e.g., a target molecule) that is recognized by an antibody or other antigen-binding moiety described herein. Epitopes can be linear or conformational.

[0027] Fab: The term "Fab" in the context of the targeting moiety of the present disclosure refers to a pair of polypeptide chains, where the first polypeptide chain comprises an antibody N-terminal variable heavy chain (VH) domain through a first constant domain (referred to herein as C1), and the second polypeptide chain comprises an antibody N-terminal variable light chain (VL) domain through a second constant domain (referred to herein as C2) that can pair with the first constant domain. In a native antibody, the VH is N-terminal to the first constant domain (CH1) of the heavy chain, and the VL is N-terminal to the constant domain (CL) of the light chain. The Fabs of the present disclosure can be oriented according to their natural orientation or can include domain substitutions or swaps that promote correct VH and VL pairing. For example, the CH1 and CL domain pair in the Fab can be replaced with a CH3 domain pair to promote modified correct Fab-chain pairing in a heterodimeric molecule. It is also possible to reverse the CH1 and CL so that CH1 is attached to VL and CL is attached to VH, a configuration commonly known as a crossmab.

[0028] Fc domain and Fc region: The term "Fc domain" refers to the portion of a heavy chain that pairs with the corresponding portion of another heavy chain. In some embodiments, an Fc domain comprises a CH2 domain followed by a CH3 domain, with or without a hinge region N-terminal to the CH2 domain. The term "Fc region" refers to the region formed by the association of two heavy chain Fc domains. The two Fc domains within an Fc region may be identical or different from each other. In natural antibodies, the Fc domains are typically identical, but one or both Fc domains may be modified to allow heterodimerization, for example, via knob-in-hole interactions.

[0029] Half antibody: The term "half antibody" refers to a molecule that contains at least one Fc domain and can associate with another molecule that contains an Fc domain, for example, through a disulfide bridge or molecular interaction. A half antibody can be composed of one polypeptide chain or two or more polypeptide chains (e.g., the two polypeptide chains of a Fab or a pMHC complex whose components are on different polypeptide chains). An example of a half antibody is a molecule that contains the heavy and light chains of an antibody (e.g., an IgG antibody). Another example of a half antibody is a molecule that contains a first polypeptide containing a VL domain and a CL domain, and a second polypeptide containing a VH domain, a CH1 domain, a hinge domain, a CH2 domain, and a CH3 domain, where the VL domain and the VH domain form an ABD. Another example of a half antibody is a polypeptide that contains an scFv domain, a CH2 domain, and a CH3 domain. Another example of a half antibody is a polypeptide that contains a pMHC complex, a CH2 domain, and a CH3 domain. When multimerized via the Fc domain, the T cell activator of the present disclosure typically contains two half antibodies. As shown in Figures 1A-1I, each half antibody typically comprises a pMHC complex and an Fc domain, or an immune cell antigen targeting moiety and an Fc domain. Each half antibody may comprise additional components, such as linkers, additional pMHC complex or immune cell antigen targeting moieties, and TAA targeting moieties, either interspersed among the above-listed components or interspersed at the ends of the half antibody.

[0030] The term "half antibody" is intended for descriptive purposes only and does not imply a particular composition or method of production. Description of half antibodies as a "first" half antibody, a "second" half antibody, a "left" half antibody, a "right" half antibody, etc. is for convenience and descriptive purposes only.

[0031] Host cell: As used herein, the term "host cell" refers to a cell into which a nucleic acid of the present disclosure has been introduced. The terms "host cell" and "recombinant host cell" are used interchangeably herein. It is understood that such terms refer to the particular subject cell and also to the progeny or potential progeny of such a cell. Because certain modifications may occur over time, either due to mutation or environmental influences, such progeny may not actually be identical to the parent cell, but are still within the scope of the term as used herein. Typical host cells are eukaryotic host cells, such as mammalian host cells. Exemplary eukaryotic host cells include yeast and mammalian cells, e.g., vertebrate cells such as mouse, rat, monkey, or human cell lines, e.g., HKB11 cells, PER.C6 cells, HEK cells, or CHO cells.

[0032] Immune cell antigen: As used herein, the term "immune cell antigen" refers to any biological molecule (e.g., a protein, carbohydrate, lipid, or a combination thereof) present on and / or expressed by an immune cell. In some embodiments, at least a portion of the immune cell antigen is extracellular (e.g., a cell surface protein or a transmembrane protein having at least one extracellular domain). Immune cells include, but are not limited to, T cells, B cells, dendritic cells (DCs), macrophages, and natural killer (NK) cells. Specific immune cell antigens contemplated herein include, but are not limited to, T cell antigens (e.g., CD3, CD28) and B cell antigens (e.g., CD20, CD22, CD19).

[0033] Immune response: The term "immune response" refers to the integrated body's response to an antigen, preferably a cellular immune response or a cellular and humoral immune response. The immune response may be protective (also "preventive" or "prophylactic") and / or therapeutic. The terms "inducing an immune response," "eliciting an immune response," and the like, as used herein, can refer to the absence of an immune response to a specific antigen before administration of a particular composition (e.g., a T cell activator), but can also refer to the presence of a certain level of immune response to a specific antigen before such administration and the enhancement of the immune response after induction. Thus, "inducing an immune response" also includes "enhancing an immune response." Preferably, after inducing an immune response in a subject, the subject is protected from developing a disease (e.g., cancer), or the disease state is improved by inducing an immune response (e.g., tumor reduction, reduction in the number of cancer cells, etc.). For example, an immune response to a tumor antigen can be induced in a patient with cancer or a subject at risk of developing cancer. Inducing an immune response in this case can mean that the subject's disease state is improved, that the subject does not develop metastases, or that a subject at risk of developing cancer does not develop cancer.

[0034] K D :「K D The term "(M)" refers to the dissociation equilibrium constant of a particular antibody-antigen interaction or the dissociation equilibrium constant of an antibody or antibody-binding fragment binding to an antigen. D There is an inverse relationship between the binding affinity and the K D The smaller the value, the higher, i.e., stronger, the affinity. Thus, the terms "higher affinity" or "stronger affinity" refer to a higher ability to form an interaction, i.e., a smaller K D Conversely, the terms "lower affinity" or "weaker affinity" refer to a lower ability to form an interaction, i.e., a larger K DIn some situations, a higher binding affinity (or K ) of a particular molecule (e.g., an antibody) to its interaction partner molecule (e.g., antigen X) compared to the binding affinity of the molecule (e.g., an antibody) to another interaction partner molecule (e.g., antigen Y) may be observed. D ) is larger than K D The smaller the K value (lower, or weaker, affinity) D The binding affinity may be expressed as a binding ratio determined by dividing by the affinity (higher, or stronger, affinity), for example, as a 5-fold or 10-fold higher binding affinity, as the case may be.

[0035] Major histocompatibility complex and MHC: These terms refer to naturally occurring MHC molecules, individual chains of MHC molecules (e.g., MHC class I α (heavy) chain, β2 microglobulin, MHC class II α chain, and MHC class II β chain), individual subunits of such chains of MHC molecules (e.g., α1, α2, and / or α3 subunits of the MHC class I α chain, α1-α2 subunits of the MHC class II α chain, and β1-β2 subunits of the MHC class II β chain), as well as portions (e.g., peptide-binding portions, e.g., peptide-binding grooves), variants, and various derivatives (including fusion proteins) thereof, which retain the ability to present antigenic peptides for recognition by T cell receptors (TCRs), e.g., antigen-specific TCRs. MHC class I molecules contain a peptide-binding groove formed by the α1 and α2 domains of the heavy α chain, which can accommodate peptides of approximately 8-10 amino acids. Despite the fact that both classes of MHC bind to a core of approximately nine amino acids (e.g., 5-17 amino acids) within a peptide, the MHC class II peptide-binding groove (the α1 domain of a class II MHC a polypeptide associated with the β1 domain of a class II MHC β polypeptide) allows for a wider range of peptide lengths. Peptides that bind to MHC class II typically vary between 13-17 amino acids in length, although shorter or longer lengths are not uncommon. As a result, peptides may shift within the MHC class II peptide-binding groove, altering which 9-mer is directly positioned within the groove at any given time. Conventional identification of specific MHC variants is used herein. This term encompasses "human leukocyte antigen" or "HLA."

[0036] Monovalent: As used herein with respect to a pMHC complex and / or targeting moiety in a T cell activator, the term "monovalent" refers to a T cell activator that has only a single pMHC complex and / or targeting moiety, respectively. In some embodiments, a T cell activator that is monovalent with respect to a pMHC complex and / or targeting moiety is a heterodimer.

[0037] Operably linked: As used herein, the term "operably linked" refers to a functional relationship between two or more regions of a polypeptide chain, where the two or more regions are joined to produce a functional polypeptide or two or more nucleic acid sequences, e.g., to produce an in-frame fusion of two polypeptide components or to link a regulatory sequence to a coding sequence.

[0038] Peptide-MHC complex, pMHC complex, peptide-in-groove: The terms "peptide-MHC complex," "pMHC complex," and "peptide-in-groove," used interchangeably herein, refer to a molecule comprising (i) an MHC domain (e.g., a human MHC molecule or portion thereof (e.g., its peptide-binding groove and, e.g., its extracellular portion)), (ii) an antigenic peptide, and, optionally, (iii) a β2 microglobulin domain (e.g., human β2 microglobulin or a portion thereof), wherein the MHC domain, antigenic peptide, and optional β2 microglobulin domain form a complex to enable specific binding to a T cell receptor. In some embodiments, a pMHC complex comprises at least the extracellular domain of a human HLA class I / human β2 microglobulin molecule and / or a human HLA class II molecule. A pMHC complex can exist as an isolated molecule or as a component of a molecule comprising one or more additional elements, e.g., as a component of a T cell activator. The pMHC complex present as a component of the T cell activator of the present disclosure is also referred to herein as the "pMHC moiety."

[0039] Prevent: The terms "prevent," "preventing," "prevention," "prophylactic treatment," and the like refer to reducing the likelihood of developing or delaying the onset of a disorder, disease, or condition in a subject who does not have the disorder, disease, or condition but is at risk of or susceptible to developing it. Prevention and the like does not solely mean preventing a subject from ever suffering from a particular disorder, disease, or condition. Prevention may require the administration of multiple doses of a treatment (e.g., a pharmaceutical composition comprising a T cell activator) disclosed herein. Prevention can include preventing the recurrence of disease in a subject in whom all disease symptoms have been eliminated (e.g., a subject in remission). Thus, as described herein, a subject "not having" a disorder or condition includes a subject who previously had the disease and in whom all disease symptoms have been eliminated. For example, a method for preventing cancer in a subject includes a method for preventing cancer in a subject who has never had cancer, and also includes a method for preventing recurrence or relapse in a subject who previously had cancer.

[0040] Protein, Peptide, and Polypeptide: The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues.

[0041] Single-chain Fv or scFv: As used herein, the term "single-chain Fv" or "scFv" refers to a polypeptide chain comprising the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain.

[0042] Specifically (or selectively) binds: The term "specifically (or selectively) binds" to an antigen or epitope refers to a binding reaction that determines the presence of the cognate antigen or epitope in a heterogeneous population of proteins and other molecules. The binding reaction can be, but need not be, mediated by an antibody or antibody fragment. The term "specifically binds" does not exclude cross-species reactivity. For example, an antigen-binding domain (e.g., an antigen-binding fragment of an antibody) that "specifically binds" to an antigen from one species may also "specifically bind" to that antigen in one or more other species. Thus, such cross-species reactivity does not in itself alter the classification of the antigen-binding domain as a "specific" binder. In certain embodiments, an antigen-binding domain of the present disclosure that specifically binds to a human antigen has cross-species reactivity with one or more non-human mammalian species, e.g., primate species (including, but not limited to, one or more of Macaca fascicularis, Macaca mulatta, and Macaca nemestrina), or rodent species, e.g., Mus musculus.

[0043] Subject: The term "subject" includes human and non-human animals. Non-human animals include all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cows, chickens, amphibians, and reptiles. Except where noted, the terms "patient" and "subject" are used interchangeably herein.

[0044] Target molecule: As used herein, the term "target molecule" refers to any biomolecule (e.g., a protein, carbohydrate, lipid, or combination thereof) that can be specifically bound by the targeting moiety of the T cell activator of the present disclosure.

[0045] T cell activator: As used herein, the term "T cell activator" refers to a molecule comprising at least one peptide-MHC complex and at least one immune cell antigen targeting moiety, e.g., at least one TCA targeting moiety and / or at least one BCA targeting moiety. Generally, a T cell activator is a molecule composed of one or more polypeptide chains (e.g., one, two, three, or four polypeptide chains) that together comprise at least one peptide-MHC complex and at least one immune cell antigen targeting moiety. The term "T cell activator," unless otherwise indicated by context, should also be understood to extend to molecules that include additional features, such as one or more multimerization moieties, one or more linker moieties, one or more tumor-associated antigen (TAA) targeting moieties, and any combination of the foregoing. The use of the term "T cell activator" is intended for convenience and descriptive purposes only and does not imply any particular activity or functional property.

[0046] T cell antigen: As used herein, the term "T cell antigen" refers to any biological molecule (e.g., a protein, carbohydrate, lipid, or a combination thereof) present on and / or expressed by a T cell. In some embodiments, at least a portion of the T cell antigen is extracellular (e.g., a cell surface protein or a transmembrane protein having at least one extracellular domain). Specific T cell antigens contemplated herein include, but are not limited to, CD3 and CD28.

[0047] Targeting moiety: As used herein, the term "targeting moiety" refers to any molecule or binding portion thereof that can specifically bind to an antigen. In certain embodiments, a targeting moiety binds to a region of a protein antigen that is extracellular (e.g., an extracellular domain on the cell surface or a transmembrane protein expressed by a cell). Exemplary targeting moieties include, but are not limited to, antibodies and antigen-binding portions thereof (e.g., Fab, scFv, etc.). A targeting moiety may be described in terms of the antigen to which it specifically binds. Thus, for example, a "T cell antigen targeting moiety" (or "TCA targeting moiety") refers to a molecule or binding portion thereof that can specifically bind to a T cell antigen (e.g., CD3, CD28, etc.). A TCA targeting moiety can also have functional activity in addition to binding to a T cell antigen. For example, a TCA targeting moiety that is a CD3 targeting moiety (e.g., an anti-CD3 antibody or antigen-binding portion thereof) can promote the clustering and activation of CD3 on the surface of T cells, while a TCA targeting moiety that is a CD28 targeting moiety (e.g., an anti-CD28 antibody or antigen-binding portion thereof) can activate CD28 signaling within T cells. Similarly, a "TCR targeting moiety" refers to a molecule or binding portion thereof that can specifically bind to a T cell receptor, and a "B cell antigen targeting moiety" (or "BCA targeting moiety") refers to a molecule or binding portion thereof that can specifically bind to a B cell antigen (e.g., CD19, CD20, CD22, etc.).

[0048] Treat, Treatment, Treating: As used herein, the terms "treat," "treatment," and "treating" refer to the shortening or amelioration of the progression, severity, and / or duration of a disorder (e.g., a proliferative disorder), or the amelioration of one or more symptoms (preferably one or more discernible symptoms) of a disorder, resulting from the administration of one or more T cell activating agents of the disclosure.

[0049] In certain embodiments, in the context of treating a proliferative disorder, the terms "treat," "treatment," and "treating" refer to an improvement in at least one measurable physical parameter of the proliferative disorder, such as tumor growth, not necessarily discernible by the patient. In other embodiments, the terms "treat," "treatment," and "treating" refer to an inhibition of progression of the proliferative disorder, either physical, e.g., by stabilization of a discernible symptom, physiological, e.g., by stabilization of a physical parameter, or both. In other embodiments, the terms "treat," "treatment," and "treating" refer to a reduction or stabilization of tumor size or cancerous cell number.

[0050] Tumor: The term "tumor" is used interchangeably herein with the term "cancer," e.g., both terms encompass solid and liquid, e.g., diffuse or circulating, tumors. As used herein, the term "cancer" or "tumor" includes precancerous and malignant cancers and tumors.

[0051] Tumor-associated antigen: The term "tumor-associated antigen" (also "tumor-associated antigen") or "TAA" refers to a molecule (typically a protein, carbohydrate, lipid, or some combination thereof) that is expressed on the surface of cancer cells, either in whole or as fragments (e.g., MHC / peptides), and is useful for preferential targeting of pharmacological agents to cancer cells. In some embodiments, a TAA is a marker expressed by both normal and cancer cells, e.g., a lineage marker, e.g., CD19 on B cells. In some embodiments, a TAA is a cell surface molecule that is overexpressed in cancer cells compared to normal cells, e.g., by 1-fold overexpression, 2-fold overexpression, 3-fold overexpression, or more compared to normal cells. In some embodiments, a TAA is a cell surface molecule that is inappropriately synthesized in cancer cells, e.g., a molecule that contains a deletion, addition, or mutation compared to the molecule expressed on normal cells. In some embodiments, a TAA is expressed exclusively on the cell surface of cancer cells, either in whole or as fragments (e.g., MHC / peptides), and is not synthesized or expressed on the surface of normal cells. Thus, the term "TAA" encompasses antigens specific to cancer cells, sometimes known in the art as tumor-specific antigens ("TSAs").

[0052] Universal light chain: As used herein in connection with a targeting moiety, the term "universal light chain" refers to a light chain polypeptide that can pair with the heavy chain region of the targeting moiety and can also pair with other heavy chain regions. A universal light chain is also known as a "common light chain."

[0053] VH: The term "VH" refers to the variable region of an immunoglobulin heavy chain of an antibody, including the heavy chain of an scFv or Fab. VL: The term "VL" refers to the variable region of an immunoglobulin light chain, including the light chain of an scFv or Fab.

[0054] 6.2. T cell activators The present disclosure provides multispecific molecules comprising a pMHC complex, one or more immune cell antigen targeting moieties (e.g., a TCA targeting moiety and / or a BCA targeting moiety), and an optional multimerization moiety. Such multispecific molecules are often referred to herein as "T cell activators." The use of the term "T cell activator" is intended for convenience and descriptive purposes only and does not imply any particular activity or functional property.

[0055] In various embodiments, the T cell activator is (a) monovalent or bivalent with respect to the pMHC complex, and / or (b) monovalent or bivalent with respect to the immune cell antigen targeting moiety. Thus, in some embodiments, the T cell activator of the present disclosure is monovalent with respect to the pMHC complex and monovalent with respect to the immune cell antigen targeting moiety. In other embodiments, the T cell activator of the present disclosure is bivalent with respect to the pMHC complex and monovalent with respect to the immune cell antigen targeting moiety. In other embodiments, the T cell activator of the present disclosure is monovalent with respect to the pMHC complex and bivalent with respect to the immune cell antigen targeting moiety. In yet further embodiments, the T cell activator of the present disclosure is bivalent with respect to the pMHC complex and bivalent with respect to the immune cell antigen targeting moiety.

[0056] Exemplary pMHC complexes suitable for use in the T cell activators of the present disclosure are described in Section 6.3. Exemplary immune cell antigen targeting moieties (including TCA and BCA targeting moieties) for use in the T cell activators of the present disclosure are described in Section 6.4.

[0057] The T cell activator can be a fusion protein comprising a pMHC complex, an immune cell antigen targeting moiety, and optionally a multimerization moiety. Exemplary multimerization moieties are described in Section 6.7 and include an Fc domain that confers homodimerization or heterodimerization capabilities to the T cell activator.

[0058] Furthermore, each of the pMHC complex, the TCA targeting moiety, and the multimerization moiety can itself be a fusion protein, for example, a pMHC complex can be a single fusion polypeptide comprising an antigenic peptide, a linker, an optional β2-microglobulin domain, an optional additional linker, and an MHC domain.

[0059] The T cell activator can include one or more linker sequences that connect various components of the molecule, e.g., connect different portions of the pMHC complex, connect the TCA targeting moiety to the multimerization moiety, or connect the pMHC complex to the multimerization moiety. Exemplary linkers are described in Section 6.8.

[0060] In certain aspects, the T cell activators of the present disclosure, after administration to a subject (e.g., a patient having or at risk of developing cancer), increase activation of T cells specific for an antigenic peptide in a pMHC complex.

[0061] Below are some exemplary orientations of the disclosed T cell activators or their individual polypeptide chains, in the N-terminal to C-terminal orientation: (1) Orientation 1: pMHC complex-optional linker-multimerization moiety-optional linker-immune cell antigen targeting moiety (or component thereof).

[0062] (2) Orientation 2: immune cell antigen targeting moiety (or component thereof)-optional linker-multimerization moiety-optional linker-pMHC complex. (3) Orientation 3: pMHC complex-optional linker-immune cell antigen targeting moiety (or component thereof)-optional linker-multimerization moiety.

[0063] (4) Orientation 4: Heterodimer containing two polypeptides, A and B: Polypeptide A:pMHC complex-optional linker-multimerization moiety, and Polypeptide B: immune cell antigen targeting moiety (or component thereof) - optional linker - multimerization moiety.

[0064] (5) Orientation 5: Heterodimer containing two polypeptides, A and B: Polypeptide A:pMHC complex-optional linker-multimerization moiety, and Polypeptide B: pMHC complex-optional linker-multimerization moiety-optional linker-immune cell antigen targeting moiety (or component thereof).

[0065] (6) Orientation 6: A homodimer or heterodimer comprising two polypeptides, A and B: Polypeptide A:pMHC complex-optional linker-multimerization moiety-optional linker-immune cell antigen targeting moiety (or a component thereof), and Polypeptide B: pMHC complex-optional linker-multimerization moiety-optional linker-immune cell antigen targeting moiety (or component thereof).

[0066] (7) Orientation 7: A homodimer or heterodimer comprising two polypeptides, A and B: Polypeptide A:pMHC complex-optional linker-immune cell antigen targeting moiety (or component thereof)-optional linker-multimerization moiety, and Polypeptide B: pMHC complex-optional linker-immune cell antigen targeting moiety (or component thereof)-optional linker-multimerization moiety.

[0067] (8) Orientation 8: Heterodimer containing two polypeptides, A and B: Polypeptide A:pMHC complex-optional linker-multimerization moiety, Polypeptide B: immune cell antigen targeting moiety (or component thereof)-optional linker-multimerization moiety-optional linker-immune cell antigen targeting moiety (or component thereof).

[0068] (9) Orientation 9: Heterodimer containing two polypeptides, A and B: Polypeptide A:pMHC complex-optional linker-multimerization moiety-optional linker-immune cell targeting moiety (or a component thereof), Polypeptide B: immune cell antigen targeting moiety (or component thereof) - optional linker - multimerization moiety.

[0069] (10) Orientation 10: A heterodimer comprising two polypeptides, A and B: Polypeptide A:pMHC complex-optional linker-multimerization moiety-optional linker-immune cell targeting moiety (or a component thereof), Polypeptide B: immune cell antigen targeting moiety (or component thereof)-optional linker-multimerization moiety-optional linker-immune cell antigen targeting moiety (or component thereof).

[0070] (11) Orientation 11: A heterodimer comprising two polypeptides, A and B: Polypeptide A:pMHC complex-optional linker-multimerization moiety, Polypeptide B: immune cell antigen targeting moiety (or component thereof)-optional linker-multimerization moiety-optional linker-tumor antigen targeting moiety (or component thereof).

[0071] (12) Orientation 12: Heterodimer containing two polypeptides, A and B: Polypeptide A:pMHC complex-optional linker-multimerization moiety-optional linker-immune cell targeting moiety (or a component thereof), Polypeptide B: tumor antigen targeting moiety (or component thereof)-optional linker-multimerization moiety-optional linker-tumor antigen targeting moiety (or component thereof).

[0072] In the foregoing embodiments, the multimerization moiety can be an Fc domain. Thus, a T cell activator that dimerizes via an Fc domain can be described as being composed of two half antibodies, and heterodimeric pairing can be achieved by pairing of the Fc heterodimerization variants described in Section 6.7.1.2.

[0073] In certain embodiments, a T cell activator of the present disclosure comprises two or more immune cell antigen targeting moieties, e.g., a T cell activator of Orientation 5, Orientation 6, Orientation 7, Orientation 8, Orientation 9, or Orientation 10. The two or more immune cell antigen targeting moieties may be the same type of immune cell antigen targeting moiety (e.g., two or more TCA targeting moieties, two or more BCA targeting moieties, etc.). A T cell activator comprising two or more TCA targeting moieties may comprise two moieties that target the same antigen (e.g., two CD3 targeting moieties or two CD28 targeting moieties) or different antigens (e.g., one CD3 targeting moiety and one CD28 targeting moiety). A T cell activator comprising two or more BCA targeting moieties may comprise two moieties that target the same antigen (e.g., two CD19 targeting moieties, two CD20 targeting moieties, or two CD22 targeting moieties) or different antigens (e.g., one CD19 targeting moiety and one CD22 targeting moiety).

[0074] Alternatively, the two or more immune cell antigen targeting moieties may comprise different types of immune cell antigen targeting moieties (e.g., one TCA targeting moiety and one BCA targeting moiety, two TCA targeting moieties and one BCA targeting moiety, two BCA targeting moieties and one TCA targeting moiety, etc.). In some embodiments, the T cell activator comprises one CD3 targeting moiety and one CD19 targeting moiety. In some embodiments, the T cell activator comprises one CD3 targeting moiety and one CD20 targeting moiety. In some embodiments, the T cell activator comprises one CD3 targeting moiety and one CD22 targeting moiety. In some embodiments, the T cell activator comprises one CD28 targeting moiety and one CD19 targeting moiety. In some embodiments, the T cell activator comprises one CD28 targeting moiety and one CD20 targeting moiety. In some embodiments, the T cell activator comprises one CD28 targeting moiety and one CD22 targeting moiety. In some embodiments, the T cell activator comprises two CD3 targeting moieties and one CD19 targeting moiety. In some embodiments, the T cell activator comprises two CD3 targeting moieties and one CD20 targeting moiety. In some embodiments, the T cell activator comprises two CD3 targeting moieties and one CD22 targeting moiety. In some embodiments, the T cell activator comprises two CD28 targeting moieties and one CD19 targeting moiety. In some embodiments, the T cell activator comprises two CD28 targeting moieties and one CD20 targeting moiety. In some embodiments, the T cell activator comprises two CD28 targeting moieties and one CD22 targeting moiety. In some embodiments, the T cell activator comprises one CD3 targeting moiety and two CD19 targeting moieties. In some embodiments, the T cell activator comprises one CD3 targeting moiety and two CD20 targeting moieties. In some embodiments, the T cell activator comprises one CD3 targeting moiety and two CD22 targeting moieties. In some embodiments, the T cell activator comprises one CD28 targeting moiety and two CD19 targeting moieties.In some embodiments, the T cell activator comprises one CD28 targeting moiety and two CD20 targeting moieties, hi some embodiments, the T cell activator comprises one CD28 targeting moiety and two CD22 targeting moieties.

[0075] An exemplary T cell activator in orientation 4 is shown in Figure 1A. The immune cell antigen targeting moiety (3) of the exemplary T cell activator shown in Figure 1A may be a TCA targeting moiety (e.g., a CD3 targeting moiety or a CD28 targeting moiety) or a BCA targeting moiety (e.g., a CD19 targeting moiety, a CD20 targeting moiety, or a CD22 targeting moiety).

[0076] An exemplary T cell activator of orientation 5 is shown in Figure 1B. The immune cell antigen targeting moiety (3) of the exemplary T cell activator shown in Figure 1B may be a TCA targeting moiety (e.g., a CD3 targeting moiety or a CD28 targeting moiety) or a BCA targeting moiety (e.g., a CD19 targeting moiety, a CD20 targeting moiety, or a CD22 targeting moiety).

[0077] An exemplary T cell activator of orientation 6 is shown in Figure 1C. The immune cell antigen targeting moiety (3) of the exemplary T cell activator shown in Figure 1C may be both a TCA targeting moiety (e.g., a CD3 targeting moiety or a CD28 targeting moiety), may be both a BCA targeting moiety (e.g., a CD19 targeting moiety, a CD20 targeting moiety, or a CD22 targeting moiety), or may be one TCA targeting moiety and one BCA targeting moiety (e.g., one CD28 targeting moiety and one CD20 targeting moiety).

[0078] An exemplary T cell activator in orientation 7 is shown in Figure ID. The immune cell antigen targeting moiety (3) of the exemplary T cell activator shown in Figure ID may be both a TCA targeting moiety (e.g., a CD3 targeting moiety or a CD28 targeting moiety), may be both a BCA targeting moiety (e.g., a CD19 targeting moiety, a CD20 targeting moiety, or a CD22 targeting moiety), or may be one TCA targeting moiety and one BCA targeting moiety (e.g., one CD28 targeting moiety and one CD20 targeting moiety).

[0079] An exemplary T cell activator of orientation 8 is shown in Figure IE. The immune cell antigen targeting moiety (3) of the exemplary T cell activator shown in Figure IE may be both a TCA targeting moiety (e.g., a CD3 targeting moiety or a CD28 targeting moiety), may be both a BCA targeting moiety (e.g., a CD19 targeting moiety, a CD20 targeting moiety, or a CD22 targeting moiety), or may be one TCA targeting moiety and one BCA targeting moiety (e.g., one CD28 targeting moiety and one CD20 targeting moiety).

[0080] An exemplary T cell activator in orientation 9 is shown in Figure IF. The immune cell antigen targeting moiety (3) of the exemplary T cell activator shown in Figure IF may be both a TCA targeting moiety (e.g., a CD3 targeting moiety or a CD28 targeting moiety), may be both a BCA targeting moiety (e.g., a CD19 targeting moiety, a CD20 targeting moiety, or a CD22 targeting moiety), or may be one TCA targeting moiety and one BCA targeting moiety (e.g., one CD28 targeting moiety and one CD20 targeting moiety).

[0081] An exemplary T cell activator of orientation 10 is shown in Figure 1G. The immune cell antigen targeting moiety (3) of the exemplary T cell activator shown in Figure 1G may be both a TCA targeting moiety (e.g., a CD3 targeting moiety or a CD28 targeting moiety), may be both a BCA targeting moiety (e.g., a CD19 targeting moiety, a CD20 targeting moiety, or a CD22 targeting moiety), or may be one TCA targeting moiety and one BCA targeting moiety (e.g., one CD28 targeting moiety and one CD20 targeting moiety).

[0082] In the T cell activators of the present disclosure, when the immune cell antigen targeting moiety is an antibody antigen binding domain ("ABD"), each immune cell antigen targeting moiety can be composed of two polypeptide chains, one polypeptide chain having a heavy chain variable region and the other polypeptide chain having a light chain variable region. Thus, the immune cell antigen targeting moiety itself can comprise heavy and light chain variable domains on separate polypeptide chains. For example, a polypeptide comprising an immune cell antigen targeting moiety can be composed of two polypeptide chains, one chain comprising the heavy chain variable domain of the targeting moiety-optional linker-multimerization moiety, and the other chain comprising the light chain variable domain of the targeting moiety.

[0083] Alternatively, an scFv can be used in which the heavy and light chain variable regions are fused to each other in a single polypeptide. When the T cell activators of the present disclosure dimerize via the Fc domain, they can be said to be composed of two half antibodies. Below are some exemplary configurations of half antibodies that can be incorporated into the T cell activators of the present disclosure.

[0084] (1) Half antibody 1: A first polypeptide chain comprising a pMHC complex-optional linker A-Fc domain-optional linker B-immune cell antigen targeting moiety or a component thereof, optionally associated with a second polypeptide chain comprising other components of the immune cell antigen targeting moiety (e.g., the first polypeptide chain may comprise the VH and CH1 of the immune cell antigen targeting Fab, and the second polypeptide chain may comprise the VL and CL of the immune cell antigen targeting Fab, or vice versa).

[0085] (2) Half antibody 2: A first polypeptide chain comprising an immune cell antigen targeting moiety or a component thereof-optional linker A-Fc domain-optional linker B-pMHC complex, optionally associated with a second polypeptide chain comprising other components of the immune cell antigen targeting moiety (e.g., the first polypeptide chain may comprise the VH and CH1 of the immune cell antigen that targets the Fab, and the second polypeptide chain may comprise the VL and CL of the immune cell antigen that targets the Fab, or vice versa).

[0086] (3) Half antibody 3: a first polypeptide chain comprising a pMHC complex-optional linker A-immune cell antigen targeting moiety or component thereof-optional linker B-Fc domain, optionally associated with a second polypeptide chain comprising other components of the immune cell antigen targeting moiety (e.g., the first polypeptide chain may comprise the VH and CH1 of the immune cell antigen targeting Fab, and the second polypeptide chain may comprise the VL and CL of the immune cell antigen targeting Fab, or vice versa).

[0087] (4) Half antibody 4: pMHC complex - optional linker A - polypeptide chain comprising an Fc domain. (5) Half antibody 5: A first polypeptide chain comprising an immune cell antigen targeting moiety or component thereof—optional linker A—Fc domain, optionally associated with a second polypeptide chain comprising other components of the immune cell antigen targeting moiety (e.g., the first polypeptide chain may comprise the VH and CH1 of the immune cell antigen that targets the Fab, and the second polypeptide chain may comprise the VL and CL of the immune cell antigen that targets the Fab, or vice versa).

[0088] (6) Half antibody 6: A first polypeptide chain comprising a first immune cell antigen targeting moiety or a component thereof—optional linker A—Fc domain—optional linker B—second immune cell antigen targeting moiety or a component thereof, optionally associated with a second polypeptide chain comprising other components of the first immune cell antigen targeting moiety (e.g., the first polypeptide chain may comprise the VH and CH1 of the immune cell antigen targeting the Fab, and the second polypeptide chain may comprise the VL and CL of the immune cell antigen targeting the Fab, or vice versa), and / or a third polypeptide chain comprising other components of the second immune cell antigen targeting moiety (e.g., the first polypeptide chain may comprise the VH and CH1 of the immune cell antigen targeting the Fab, and the third polypeptide chain may comprise the VL and CL of the immune cell antigen targeting the Fab, or vice versa).

[0089] (7) Half antibody 7: A first polypeptide chain comprising an immune cell antigen targeting moiety or a component thereof—optional linker A—Fc domain—optional linker B—tumor antigen targeting moiety or a component thereof, optionally associated with a second polypeptide chain comprising other components of the immune cell antigen targeting moiety (e.g., the first polypeptide chain may comprise the VH and CH1 of the immune cell antigen targeting the Fab, and the second polypeptide chain may comprise the VL and CL of the immune cell antigen targeting the Fab, or vice versa), and / or a third polypeptide chain comprising other components of the tumor antigen targeting moiety (e.g., the first polypeptide chain may comprise the VH and CH1 of the tumor antigen targeting the Fab, and the third polypeptide chain may comprise the VL and CL of the tumor antigen targeting the Fab, or vice versa).

[0090] (8) Half antibody 8: A first polypeptide chain comprising a tumor antigen targeting moiety or a component thereof—optional linker A—Fc domain—optional linker B—tumor antigen targeting moiety or a component thereof, optionally associated with a second polypeptide chain comprising other components of the tumor antigen targeting moiety (e.g., the first polypeptide chain may comprise the VH and CH1 of a tumor antigen targeting the Fab, and the second polypeptide chain may comprise the VL and CL of a tumor antigen targeting the Fab, or vice versa), and / or a third polypeptide chain comprising other components of the tumor antigen targeting moiety (e.g., the first polypeptide chain may comprise the VH and CH1 of a tumor antigen targeting the Fab, and the third polypeptide chain may comprise the VL and CL of a tumor antigen targeting the Fab, or vice versa).

[0091] In some embodiments, the T cell activator of the present disclosure comprises or consists of half antibody 5 and half antibody 4. One embodiment of this T cell activator is shown in Figure 1A, with half antibody 5 on the left and half antibody 4 on the right.

[0092] In some embodiments, the T cell activator of the present disclosure comprises or consists of half antibody 4 and half antibody 1. One embodiment of this T cell activator is shown in Figure 1B, with half antibody 4 on the left and half antibody 1 on the right.

[0093] In some embodiments, a T cell activator of the present disclosure comprises or consists of two of half antibodies 1. One embodiment of this T cell activator is shown in Figure 1C. Each half antibody 1 may comprise the same type of immune cell antigen targeting moiety (e.g., a T cell activator may comprise two TCA targeting moieties that may target the same or different TCAs, or two BCA targeting moieties that may target the same or different BCAs), or may comprise different types of immune cell antigen targeting moieties (e.g., a T cell activator may comprise one TCA targeting moiety and one BCA targeting moiety).

[0094] In some embodiments, a T cell activator of the present disclosure comprises or consists of two of the half antibodies 3. One embodiment of this T cell activator is shown in Figure ID. Each half antibody 3 may comprise the same type of immune cell antigen targeting moiety (e.g., a T cell activator may comprise two TCA targeting moieties that may target the same or different TCAs, or two BCA targeting moieties that may target the same or different BCAs), or may comprise different types of immune cell antigen targeting moieties (e.g., a T cell activator may comprise one TCA targeting moiety and one BCA targeting moiety).

[0095] In some embodiments, a T cell activator of the present disclosure comprises or consists of half antibody 4 and half antibody 6. One embodiment of this T cell activator is shown in Figure IE, with half antibody 6 on the left and half antibody 4 on the right.

[0096] In some embodiments, the T cell activator of the present disclosure comprises or consists of half antibody 1 and half antibody 5. One embodiment of this T cell activator is shown in Figure IF, with half antibody 5 on the left and half antibody 1 on the right.

[0097] In some embodiments, the T cell activator of the present disclosure comprises or consists of half antibody 1 and half antibody 6. One embodiment of this T cell activator is shown in Figure 1G, with half antibody 6 on the left and half antibody 1 on the right.

[0098] In some embodiments, a T cell activator of the present disclosure comprises or consists of half antibody 4 and half antibody 7. One embodiment of this T cell activator is shown in Figure 1H, with half antibody 7 on the left and half antibody 4 on the right.

[0099] In some embodiments, the T cell activator of the present disclosure comprises or consists of half antibody 1 and half antibody 8. One embodiment of this T cell activator is shown in Figure 1I, with half antibody 8 on the left and half antibody 1 on the right.

[0100] In some embodiments, the T cell activator of the present disclosure comprises or consists of half antibody 2 and half antibody 5. In further embodiments, the T cell activator of the present disclosure comprises or consists of two of half antibodies 2.

[0101] Heterodimeric T cell activators (e.g., as shown in Figures 1A, 1B, and 1E-1H) can associate via pairing of Fc heterodimerization variants, as described in section 6.7.1.2.

[0102] Further details of the components of the T cell activators of the present disclosure are provided below. Table A below shows certain embodiments of specific T cell activators, including the corresponding half-antibody pairs and their binding domains.

[0103] [Table 1-1]

[0104] [Table 1-2]

[0105] Table 1-3

[0106] Table 1-4

[0107] Table 1-5

[0108] Table 1-6

[0109] Table 1-7

[0110] Table 1-8

[0111] Table 1-9

[0112] Table 1-10

[0113] Table 1-11

[0114] Table 1-12

[0115] Table 1-13

[0116] Table 1-14

[0117] Table 1-15

[0118] Table 1-16

[0119] Table 1-17

[0120] Table 1-18

[0121] Table 1-19

[0122] Table 1-20

[0123] Table 1-21

[0124] Table 1-22

[0125] Table 1-23

[0126] Table 1-24

[0127] Table 1-25

[0128] Table 1-26

[0129] Table 1-27

[0130] Table 1-28

[0131] Table 1-29

[0132] Table 1-30

[0133] Table 1-31

[0134] Table 1-32

[0135] [Table 1-33]

[0136] [Table 1-34]

[0137] [Table 1-35]

[0138] [Table 1-36]

[0139] 6.3. Peptide-MHC Complexes Aspects of the present disclosure relate to molecules (e.g., T cell activators) that comprise peptide-MHC complexes ("pMHC complexes") that include a peptide complexed with an MHC class I domain or a peptide complexed with an MHC class II domain, optionally complexed with a β2 microglobulin (β2M) domain.

[0140] In some embodiments, a pMHC complex comprises (i) an antigenic peptide, (ii) a class I MHC polypeptide, or a fragment, variant, or derivative thereof (e.g., the extracellular domain), and, optionally, (iii) a β2-microglobulin polypeptide, or a fragment, variant, or derivative thereof. In the context of the T cell activators of the present disclosure, the components of a pMHC complex can be present in a single polypeptide chain. For example, a pMHC complex can comprise, from N- to C-terminus, (i) an antigenic peptide, (ii) a β2M sequence, and (iii) a class I α (heavy) chain sequence. Alternatively, a pMHC complex can comprise, from N- to C-terminus, (i) an antigenic peptide, (ii) a class I α (heavy) chain sequence, and (iii) a β2M sequence.

[0141] In certain embodiments, the antigenic peptide and MHC sequence and / or the MHC sequence and β2M domain are linked to each other via a peptide linker, e.g., as described in Section 6.8. In some embodiments, the single-chain pMHC complex can include a first flexible linker between the peptide segment and the β2 microglobulin segment. For example, the linker can extend from the carboxy terminus of the peptide and connect to the amino terminus of the β2 microglobulin segment. In some embodiments, the linker is structured to allow the peptide to fold into the binding groove and provide a functional pMHC complex. In some embodiments, the linker can include at least 3 amino acids and up to about 15 amino acids (e.g., 20 amino acids). The pMHC linker can include a second flexible linker inserted between the β2 microglobulin and the MHC I heavy chain segment. For example, the linker can extend from the carboxy terminus of the β2 microglobulin segment and connect to the amino terminus of the MHC I heavy chain segment. In certain embodiments, β2 microglobulin and an MHC I heavy chain result in a molecule that can fold into the binding groove and function in promoting T cell expansion.

[0142] In further embodiments, a single-chain pMHC complex can comprise a peptide covalently linked to an MHC class I α (heavy) chain via a disulfide bridge (i.e., a disulfide bond between two cysteines). See, e.g., U.S. Pat. Nos. 8,992,937 and 8,895,020, each of which is incorporated by reference in its entirety. In certain embodiments, the disulfide bond comprises a first cysteine ​​located within a linker extending from the carboxy terminus of the peptide and a second cysteine ​​located within the MHC class I heavy chain (e.g., the MHC class I α (heavy) chain having the noncovalent binding site for the antigenic peptide). In certain embodiments, the second cysteine ​​can be a mutation (addition or substitution) in the MHC class I α (heavy) chain. Preferably, the pMHC complex comprises one contiguous polypeptide chain and a disulfide bridge. Alternatively, the pMHC complex can comprise two contiguous polypeptide chains linked via a disulfide bridge as the only covalent bond. In some embodiments, the linking sequence can contain at least one amino acid in addition to cysteine, including one or more glycines, one or more alanines, and / or one or more serines. In some embodiments, the single-chain molecule comprises, from N- to C-terminus, an MHC class I peptide (e.g., an antigenic peptide), a first linker comprising a first cysteine, a β2-microglobulin sequence, a second linker, and an MHC class I heavy chain sequence comprising a second cysteine, wherein the first cysteine ​​and the second cysteine ​​comprise a disulfide bridge. In some embodiments, the second cysteine ​​is a substitution of an amino acid in the MHC class I heavy chain selected from the group consisting of T80C, Y84C, and N86C (Y84C refers to a mutation at position 108 in the mature protein, which lacks a signal sequence; alternatively, if the protein still contains a 24-mer signal sequence, that position is referred to instead as Y108C).

[0143] In certain embodiments, a disulfide bridge can link an antigenic peptide within the class I groove of a pMHC complex when the pMHC complex contains a first cysteine ​​in a Gly-Ser linker extending between the C-terminus of the peptide and β2 microglobulin, and a second cysteine ​​at an adjacent heavy chain position.

[0144] MHC Naturally occurring MHC is encoded by a cluster of genes on human chromosome 6. MHC includes, but is not limited to, HLA specificities such as A (e.g., A1-A74), B (e.g., B1-B77), C (e.g., C1-C11), D (e.g., D1-D26), DR (e.g., DR1-DR8), DQ (e.g., DQ1-DQ9), and DP (e.g., DP1-DP6). HLA specificities include A1, A2, A3, All, A23, A24, A28, A30, A33, B7, B8, B35, B44, B53, B60, B62, DR1, DR2, DR3, DR4, DR7, DR8, and DR11.

[0145] Naturally occurring MHC class I molecules bind peptides derived from proteolytic proteins, and the resulting small peptides are transported to the endoplasmic reticulum, where they associate with nascent MHC class I molecules, which are then routed through the Golgi apparatus and presented on the cell surface for recognition by cytotoxic T lymphocytes.

[0146] Naturally occurring MHC class I molecules consist of an α (heavy) chain associated with β2 microglobulin. The heavy chain consists of subunits α1-α3. The β2 microglobulin protein and the α3 subunit of the heavy chain are associated. In certain embodiments, the β2 microglobulin and the α3 subunit are associated by a covalent bond. In certain embodiments, the β2 microglobulin and the α3 subunit are associated non-covalently. The α1 and α2 subunits of the heavy chain fold to form a groove for peptides, e.g., antigenic determinants, to be presented and recognized by the TCR.

[0147] Class I molecules generally associate with, e.g., bind to, peptides about 8-9 amino acids in length (e.g., 7-11 amino acids). Every human has 3-6 different Class I molecules, each capable of binding many different types of peptides. In one particular embodiment, the Class I MHC polypeptide is a human Class I MHC polypeptide selected from the group consisting of HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, and HLA-G.

[0148] In some embodiments, the pMHC complex comprises an MHC class I alpha heavy chain extracellular domain (human alpha 1, alpha 2, and / or alpha 3 domains) without a transmembrane domain. In some embodiments, the class I alpha heavy chain polypeptide is HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-G, HLA-K, or HLA-L.

[0149] Non-limiting examples of HLA-A alleles include, but are not limited to, A*0101, A*0201, A*0202, A*0301, A*1101, A*2301, A*2402, A*2501, A*2601, A*2901, A*2902, A*3101, A*3201, A*3301, A*3401, A*3601, A*4301, A*6601, A*6801, A*6901, A*7401, and A*8001. Non-limiting examples of HLA-B alleles include, but are not limited to, B*0702. B*0801, B*1301, B*1401, B*1402, B*1501, B*1801, B*1802, B*2701, B*2702, B*3501, B*3502, B*3701, B*3801, B*3901, B*4001, B*4101, B*4201, B*4402, B*4501, B*4601, B *4701, B*4801, B*4901, B*5001, B*5101, B*5201, B*5301, B*5401, B*5501, B*5502, B*5601, B*5701, B*5801, B*5901, B*6701, B*7301, B*1517, B*8101, B*8201, and B*8301. Non-limiting examples of HLA-C alleles include, but are not limited to, Cw*0101, Cw*0202, Cw*0303, Cw*0401, Cw*0501, Cw*0602, Cw*0701, Cw*0702, Cw*0802, Cw*1203, Cw*1401, Cw*1502, Cw*1601, Cw*1701, and Cw*1801. Non-limiting examples of HLA-DR alleles include, but are not limited to, DRB1*0101, DRB1*0103, DRB1*1501, DRB1*1502, DRB1*1601, DRB1*1602, DRB1*0301, DRB1*0401, DRB1*0404, DRB1*1101, DRB1*1201, DRB1*1301, DRB1*1302, DRB1*1401, DRB1*1402, DRB1*0701, DRB1*0801, DRB1*0802, DRB1*0803, DRB1*0901, and DRB1*1001.In some embodiments, the MHC class I molecule can be selected from HLA-A*02, HLA-A*01, HLA-A*03, HLA-A*11, HLA-A*23, HLA-A*24, HLA-B*07, HLA-B*08, HLA-B*40, HLA-B*44, HLA-B*15, HLA-C*04, HLA*C*03, and HLA-C*07. Many allelic variants of the above HLA types are recognized in the art, and such variants are encompassed by the present disclosure. In some embodiments, the MHC molecule is HLA-A*02 or HLA-A*11.

[0150] In some embodiments, the HLA-A sequence may be the HLA-A*0201 sequence disclosed below. HLA-A*0201-GSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDGETRKVKAHSQTHRVDLGTLRGCYNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQYAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRYLENGKETLQRTDAPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQRDGEDQTQDTELVETRPAGDGTFQKWAAVVVPSGQEQRYTCHVQHEGLPKPLTLRWEP (SEQ ID NO: 251) In some embodiments, the MHC sequence has at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 251. In some embodiments, the MHC sequence has at least about 91% sequence identity to the amino acid sequence of SEQ ID NO: 251. In some embodiments, the MHC sequence has at least about 92% sequence identity to the amino acid sequence of SEQ ID NO: 251. In some embodiments, the MHC sequence has at least about 93% sequence identity to the amino acid sequence of SEQ ID NO: 251. In some embodiments, the MHC sequence has at least about 94% sequence identity to the amino acid sequence of SEQ ID NO: 251. In some embodiments, the MHC sequence has at least about 95% sequence identity to the amino acid sequence of SEQ ID NO: 251. In some embodiments, the MHC sequence has at least about 96% sequence identity to the amino acid sequence of SEQ ID NO: 251. In some embodiments, the MHC sequence has at least about 97% sequence identity to the amino acid sequence of SEQ ID NO: 251. In some embodiments, the MHC sequence has at least about 98% sequence identity to the amino acid sequence of SEQ ID NO: 251. In some embodiments, the MHC sequence has at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 251. In some embodiments, the MHC sequence has 100% sequence identity to the amino acid sequence of SEQ ID NO: 251.

[0151] As an alternative to a type I MHC-based pMHC complex, the T cell activator of the present disclosure can include a class II MHC-based pMHC complex. A class II MHC-based pMHC complex generally includes a class I MHC polypeptide or a fragment, variant, or derivative thereof. In one specific embodiment, the MHC includes the α and β polypeptides of a class II MHC molecule or a fragment, variant, or derivative thereof. In one specific embodiment, the α and β polypeptides are linked by a peptide linker. In one specific embodiment, the MHC includes the α and β polypeptides of a human class II MHC molecule selected from the group consisting of HLA-DP, HLA-DR, HLA-DQ, HLA-DM, and HLA-DO.

[0152] MHC class II molecules generally consist of two polypeptide chains, α and β. The chains can be derived from the DP, DQ, or DR gene clusters. There are approximately 40 known different human MHC class II molecules. They all have the same basic structure, but their molecular architectures vary slightly. MHC class II molecules bind peptides 13 to 18 amino acids in length.

[0153] In some embodiments, the pMHC complex comprises one or more MHC class II α chains or extracellular portions thereof, hi some embodiments, the class II α chain is HLA-DMA, HLA-DOA, HLA-DPA, HLA-DQA, or HLA-DRA.

[0154] In other embodiments, the pMHC complex comprises one or more MHC class II β chains or extracellular portions thereof, hi some embodiments, the class II β chain is HLA-DMB, HLA-DOB, HLA-DPB, HLA-DQB, or HLA-DRB.

[0155] Class II MHC-based pMHC complexes include those described in PCT Publication No. WO2021 / 113297A1, which is incorporated herein by reference.

[0156] 6.3.2. Antigenic Peptides The peptide in the pMHC complex can have an amino acid sequence of a peptide that can associate with, eg, be presented by, an MHC molecule.

[0157] In some embodiments, the peptide in the pMHC complex comprises the amino acid sequence of a peptide that associates with a class I MHC molecule. In certain embodiments, the sequence can comprise 6 to 20 contiguous amino acids. In certain embodiments, the peptide sequence can be a protein fragment, e.g., derived from a portion of a cellular protein, e.g., a protein associated with a cancer or cancer neoantigen, and the peptide can bind to an MHC class I heavy chain.

[0158] In other embodiments, the peptide in the pMHC complex comprises an amino acid sequence of a peptide that associates with, e.g., is presented by, a class II MHC molecule, e.g., as described in WO2021 / 113297A1.

[0159] The peptide in the pMHC complex can be, for example, any peptide capable of binding to an MHC protein in such a way that the pMHC complex is capable of binding to a TCR in a specific manner.

[0160] Examples include peptides produced by hydrolysis and, most typically, synthetically produced peptides, including randomly generated peptides, specifically designed peptides, and peptides in which at least some of the amino acid positions are conserved among several peptides, with the remaining positions being random.

[0161] In nature, peptides produced by hydrolysis undergo hydrolysis before antigen binding to MHC proteins. Class I MHC typically presents peptides derived from proteins actively synthesized in the cell's cytoplasm. In contrast, class II MHC typically presents peptides derived from either exogenous proteins that enter the cell's endocytic pathway or proteins synthesized in the ER. Intracellular transport allows peptides to associate with MHC proteins.

[0162] Peptide binding to the MHC peptide-binding groove can control the spatial arrangement of MHC and / or peptide amino acid residues recognized by the TCR or pMHC-binding protein produced by genetically modified animals as disclosed herein. Such spatial control is due in part to hydrogen bonds formed between the peptide and the MHC protein. Based on knowledge of how peptides bind to various MHCs, key MHC anchor amino acids and surface-exposed amino acids that differ between different peptides can be determined. In some embodiments, the length of the MHC-binding peptide is 5-40 amino acid residues, e.g., 6-30 amino acid residues, e.g., 8-20 amino acid residues, e.g., 9-11 amino acid residues, including peptides of any size from 5-40 amino acids long (i.e., 5, 6, 7, 8, 9...40), all in whole-number increments. Naturally, MHC class II-binding peptides vary from about 9-40 amino acids, and in almost all cases, peptides can be shortened to the 9-11 amino acid core without loss of MHC binding activity or T cell recognition.

[0163] Peptides in pMHC complexes for incorporation into the T cell activators of the present disclosure are typically at least a portion of a protein, e.g., an antigenic determinant, of an infectious pathogen (e.g., a bacterium, virus, or parasite), an allergen, or a tumor-associated protein. In some embodiments, the pMHC complex comprises an antigenic determinant of a cancer cell. Exemplary antigenic determinants of cancer cells are listed in Table 2-A and include LCMV-derived peptides gp33-41, APF (126-134), BALF (276-284), CEA (571-579), CMV pp65 (495-503), FLU-M1 (58-66), gp100 (154-162), gp100 (209-217), HBV Core (18-27), Her2 / neu (369-377; V2v9); HPV E7 (11-20), HPV E7 (11-19), HPV These include E7(82-90), KLK4(11-19), LMP1(125-133), MAGE-A3(112-120), MAGE-A4(230-239), MAGE-A4(286-294), NYESO1(157-165, C165A), NYESO1(157-165, C165V), p54 WT(264-272), PAP-3(136-143), PSMA(4-12), PSMA(135-145), survivin(96-104), tyrosinase(369-377, 371D), and WT1(126-134). An exemplary HPV E7(11-19) peptide sequence is YMLDLQPET (SEQ ID NO: 7), SEQ ID NO: 537, of International Publication No. WO 2019 / 005897. An exemplary HPV E7(82-90) peptide sequence is LLMGTLGIV (SEQ ID NO: 8), SEQ ID NO: 538, of International Publication No. WO 2019 / 005897. The contents of International Publication No. WO 2019 / 005897 are incorporated herein by reference in their entirety.

[0164] [Table 2]

[0165] In some embodiments, the pMHC complexes incorporated into the T cell activators of the present disclosure comprise a peptide selected from those listed in Table 1-A. Other antigenic determinants suitable for use on cancer cells in pMHC complexes incorporated into the T cell activators of the present disclosure are the cancer neoantigens and their corresponding HLA alleles listed below in Table 1-B. The neoantigens contain mutations relative to the wild-type allele or result from the expression of a new open reading frame in the cancer cell, as set forth in Table 1 of Fritsch et al., 2014, Cancer Immunol Res 2:522-529, which is incorporated herein by reference in its entirety.

[0166] [Table 3-1]

[0167] [Table 3-2]

[0168] In some embodiments, the pMHC complexes incorporated into the T cell activators of the present disclosure comprise peptides selected from those listed in Table 2-B. Additional antigenic determinants on cancer cells suitable for use in pMHC complexes incorporated into the T cell activators of the present disclosure include those described in the Cancer Epitope Database and Analysis Resource (CEDAR), accessible on the web at cedar.iedb.org, which is a subset of the Immune Epitope Database (IEDB) described in Vita et al., 2018, Nucleic Acids Res. 8;47(D1):D339-D343, which is incorporated herein by reference in its entirety.

[0169] Other antigenic determinants suitable for incorporation into the pMHC complexes of the present disclosure include those listed in Table 1-C below.

[0170] [Table 4-1]

[0171] [Table 4-2]

[0172] In some embodiments, the pMHC complexes incorporated into the T cell activators of the present disclosure comprise a peptide selected from those listed in Table 1-C. Additional antigenic determinants suitable for use in pMHC complexes incorporated into the T cell activators of the present disclosure are those disclosed in PCT Publication No. WO2021 / 003357A1 as SEQ ID NOs: 269, 270, and 291, which are incorporated herein by reference. Still additional antigenic determinants suitable for use in pMHC complexes incorporated into the T cell activators of the present disclosure are those disclosed in U.S. Patent Application Publication No. 2022 / 0409732A1 as SEQ ID NOs: 44, 45, 46, 69, 70, 71, 72, and 73, which are incorporated herein by reference. Additional antigenic determinants suitable for use in pMHC complexes incorporated into the T cell activators of the present disclosure include those disclosed in International Publication No. WO 2023 / 240085 A1, U.S. Patent Application Publication No. 2007 / 0020327 A1, and U.S. Patent Application Publication No. 2006 / 0079453 A1, the contents of which are incorporated herein by reference in their entireties. Additional antigenic determinants contemplated herein include, for example, those described in the IEDB, accessible via the web at iedb.org and described in Vita et al., 2018, Nucleic Acids Res. 8;47(D1):D339-D343.

[0173] β2-microglobulin As noted above, the pMHC complex can optionally include β2 microglobulin (β2M). When β2M is present, the pMHC complex can include mutations in the β2M and MHC class I α heavy chain domains such that a disulfide bond can form between them. Exemplary amino acid pairs that can be substituted with cysteine ​​to allow disulfide bonding between the two domains are identified in Table 2 below or described in PCT Publication No. WO2015 / 195531, which is incorporated herein by reference in its entirety.

[0174] [Table 5]

[0175] When present, the β2 microglobulin sequence can include a full-length (human or non-human) β2 microglobulin sequence. An exemplary human β2 microglobulin sequence is Genbank Accession No. AF072097.1, the amino acid sequence of which is shown below.

[0176] Human β2 microglobulin (full length) - MSRSVALAVLALLSLSGLEAIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDM (SEQ ID NO: 249) In some embodiments, the β2 microglobulin sequence has at least about 90% sequence identity to the amino acid sequence of full-length human β2 microglobulin (SEQ ID NO: 249). In some embodiments, the β2 microglobulin sequence has at least about 91% sequence identity to the amino acid sequence of full-length human β2 microglobulin (SEQ ID NO: 249). In some embodiments, the β2 microglobulin sequence has at least about 92% sequence identity to the amino acid sequence of full-length human β2 microglobulin (SEQ ID NO: 249). In some embodiments, the β2 microglobulin sequence has at least about 93% sequence identity to the amino acid sequence of full-length human β2 microglobulin (SEQ ID NO: 249). In some embodiments, the β2 microglobulin sequence has at least about 94% sequence identity to the amino acid sequence of full-length human β2 microglobulin (SEQ ID NO: 249). In some embodiments, the β2 microglobulin sequence has at least about 95% sequence identity to the amino acid sequence of full-length human β2 microglobulin (SEQ ID NO: 249). In some embodiments, the β2 microglobulin sequence has at least about 96% sequence identity to the amino acid sequence of full-length human β2 microglobulin (SEQ ID NO: 249). In some embodiments, the β2 microglobulin sequence has at least about 97% sequence identity to the amino acid sequence of full-length human β2 microglobulin (SEQ ID NO: 249). In some embodiments, the β2 microglobulin sequence has at least about 98% sequence identity to the amino acid sequence of full-length human β2 microglobulin (SEQ ID NO: 249). In some embodiments, the β2 microglobulin sequence has at least about 99% sequence identity to the amino acid sequence of full-length human β2 microglobulin (SEQ ID NO: 249). In some embodiments, the β2 microglobulin sequence has 100% sequence identity to the amino acid sequence of full-length human β2 microglobulin (SEQ ID NO: 249).

[0177] In certain embodiments, the β2 microglobulin sequence lacks a leader peptide sequence. Thus, the β2 microglobulin sequence can contain approximately 99 amino acids. An exemplary human β2 microglobulin sequence lacking a leader peptide sequence is shown below:

[0178] Human beta2 microglobulin (without leader peptide sequence) - IQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDM (SEQ ID NO: 250) In some embodiments, the β2 microglobulin sequence has at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to the amino acid sequence of human β2 microglobulin lacking the leader peptide sequence (SEQ ID NO: 250).

[0179] In some embodiments, the β2 microglobulin sequence has at least about 90% sequence identity to the amino acid sequence of human β2 microglobulin lacking a leader peptide sequence (SEQ ID NO: 250). In some embodiments, the β2 microglobulin sequence has at least about 91% sequence identity to the amino acid sequence of human β2 microglobulin lacking a leader peptide sequence (SEQ ID NO: 250). In some embodiments, the β2 microglobulin sequence has at least about 92% sequence identity to the amino acid sequence of human β2 microglobulin lacking a leader peptide sequence (SEQ ID NO: 250). In some embodiments, the β2 microglobulin sequence has at least about 93% sequence identity to the amino acid sequence of human β2 microglobulin lacking a leader peptide sequence (SEQ ID NO: 250). In some embodiments, the β2 microglobulin sequence has at least about 94% sequence identity to the amino acid sequence of human β2 microglobulin lacking a leader peptide sequence (SEQ ID NO: 250). In some embodiments, the β2 microglobulin sequence has at least about 95% sequence identity to the amino acid sequence of human β2 microglobulin lacking a leader peptide sequence (SEQ ID NO: 250). In some embodiments, the β2 microglobulin sequence has at least about 96% sequence identity to the amino acid sequence of human β2 microglobulin lacking a leader peptide sequence (SEQ ID NO: 250). In some embodiments, the β2 microglobulin sequence has at least about 97% sequence identity to the amino acid sequence of human β2 microglobulin lacking a leader peptide sequence (SEQ ID NO: 250). In some embodiments, the β2 microglobulin sequence has at least about 98% sequence identity to the amino acid sequence of human β2 microglobulin lacking a leader peptide sequence (SEQ ID NO: 250). In some embodiments, the β2 microglobulin sequence has at least about 99% sequence identity to the amino acid sequence of human β2 microglobulin lacking a leader peptide sequence (SEQ ID NO: 250). In some embodiments, the β2 microglobulin sequence has 100% sequence identity with the amino acid sequence of human β2 microglobulin lacking the leader peptide sequence (SEQ ID NO: 250).

[0180] 6.3.4 Exemplary Peptide-MHC Complexes In certain embodiments, pMHC complexes suitable for use in the T cell activators of the present disclosure include, in N-terminal to C-terminal orientation, an antigenic peptide (e.g., as described in Section 6.3.2), a first linker (optionally containing a cysteine, which can favor disulfide bonding as described above), a human β2 microglobulin sequence (e.g., as described in Section 6.3.3), a second linker, and an MHC sequence (e.g., as described in Section 6.3.1). An exemplary pMHC sequence, excluding the peptide sequence, is as follows, where the bolded regions are the first and second linkers, the italicized region is the human β2 microglobulin sequence, and the underlined region is the human HLA-A*0201 sequence:

[0181] [ka]

[0182] In some embodiments, the pMHC complex comprises an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 252. In some embodiments, the pMHC complex comprises an amino acid sequence having at least about 91% sequence identity to the amino acid sequence of SEQ ID NO: 252. In some embodiments, the pMHC complex comprises an amino acid sequence having at least about 92% sequence identity to the amino acid sequence of SEQ ID NO: 252. In some embodiments, the pMHC complex comprises an amino acid sequence having at least about 93% sequence identity to the amino acid sequence of SEQ ID NO: 252. In some embodiments, the pMHC complex comprises an amino acid sequence having at least about 94% sequence identity to the amino acid sequence of SEQ ID NO: 252. In some embodiments, the pMHC complex comprises an amino acid sequence having at least about 95% sequence identity to the amino acid sequence of SEQ ID NO: 252. In some embodiments, the pMHC complex comprises an amino acid sequence having at least about 96% sequence identity to the amino acid sequence of SEQ ID NO: 252. In some embodiments, the pMHC complex comprises an amino acid sequence having at least about 97% sequence identity to the amino acid sequence of SEQ ID NO: 252. In some embodiments, the pMHC complex comprises an amino acid sequence having at least about 98% sequence identity to the amino acid sequence of SEQ ID NO: 252. In some embodiments, the pMHC complex comprises an amino acid sequence having at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 252. In some embodiments, the pMHC complex comprises an amino acid sequence having 100% sequence identity to the amino acid sequence of SEQ ID NO: 252.

[0183] Various antigenic peptides, including those described in Section 6.3.2 and recognized in the art, may comprise a sequence directly N-terminal to a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 252. Certain exemplary pMHC complexes (including antigenic peptides) suitable for incorporation into the T cell activators of the present disclosure, along with their amino acid sequences, are set forth in Table P below.

[0184] [Table 6]

[0185] In some embodiments, the pMHC complex comprises an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 241. In some embodiments, the pMHC complex comprises an amino acid sequence having at least about 91% sequence identity to the amino acid sequence of SEQ ID NO: 241. In some embodiments, the pMHC complex comprises an amino acid sequence having at least about 92% sequence identity to the amino acid sequence of SEQ ID NO: 241. In some embodiments, the pMHC complex comprises an amino acid sequence having at least about 93% sequence identity to the amino acid sequence of SEQ ID NO: 241. In some embodiments, the pMHC complex comprises an amino acid sequence having at least about 94% sequence identity to the amino acid sequence of SEQ ID NO: 241. In some embodiments, the pMHC complex comprises an amino acid sequence having at least about 95% sequence identity to the amino acid sequence of SEQ ID NO: 241. In some embodiments, the pMHC complex comprises an amino acid sequence having at least about 96% sequence identity to the amino acid sequence of SEQ ID NO: 241. In some aspects, the pMHC complex comprises an amino acid sequence having at least about 97% sequence identity to the amino acid sequence of SEQ ID NO: 241. In some aspects, the pMHC complex comprises an amino acid sequence having at least about 98% sequence identity to the amino acid sequence of SEQ ID NO: 241. In some aspects, the pMHC complex comprises an amino acid sequence having at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 241. In some aspects, the pMHC complex comprises an amino acid sequence having 100% sequence identity to the amino acid sequence of SEQ ID NO: 241. In certain aspects of each of the foregoing embodiments, the peptide in the pMHC complex is identical to the peptide component of SEQ ID NO: 241 or has at most one or two amino acid substitutions.

[0186] In some embodiments, the pMHC complex comprises an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 243. In some embodiments, the pMHC complex comprises an amino acid sequence having at least about 91% sequence identity to the amino acid sequence of SEQ ID NO: 243. In some embodiments, the pMHC complex comprises an amino acid sequence having at least about 92% sequence identity to the amino acid sequence of SEQ ID NO: 243. In some embodiments, the pMHC complex comprises an amino acid sequence having at least about 93% sequence identity to the amino acid sequence of SEQ ID NO: 243. In some embodiments, the pMHC complex comprises an amino acid sequence having at least about 94% sequence identity to the amino acid sequence of SEQ ID NO: 243. In some embodiments, the pMHC complex comprises an amino acid sequence having at least about 95% sequence identity to the amino acid sequence of SEQ ID NO: 243. In some embodiments, the pMHC complex comprises an amino acid sequence having at least about 96% sequence identity to the amino acid sequence of SEQ ID NO: 243. In some aspects, the pMHC complex comprises an amino acid sequence having at least about 97% sequence identity to the amino acid sequence of SEQ ID NO: 243. In some aspects, the pMHC complex comprises an amino acid sequence having at least about 98% sequence identity to the amino acid sequence of SEQ ID NO: 243. In some aspects, the pMHC complex comprises an amino acid sequence having at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 243. In some aspects, the pMHC complex comprises an amino acid sequence having 100% sequence identity to the amino acid sequence of SEQ ID NO: 243. In certain aspects of each of the foregoing embodiments, the peptide in the pMHC complex is identical to the peptide component of SEQ ID NO: 243 or has at most one or two amino acid substitutions.

[0187] In some embodiments, the pMHC complex comprises an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 245. In some embodiments, the pMHC complex comprises an amino acid sequence having at least about 91% sequence identity to the amino acid sequence of SEQ ID NO: 245. In some embodiments, the pMHC complex comprises an amino acid sequence having at least about 92% sequence identity to the amino acid sequence of SEQ ID NO: 245. In some embodiments, the pMHC complex comprises an amino acid sequence having at least about 93% sequence identity to the amino acid sequence of SEQ ID NO: 245. In some embodiments, the pMHC complex comprises an amino acid sequence having at least about 94% sequence identity to the amino acid sequence of SEQ ID NO: 245. In some embodiments, the pMHC complex comprises an amino acid sequence having at least about 95% sequence identity to the amino acid sequence of SEQ ID NO: 245. In some embodiments, the pMHC complex comprises an amino acid sequence having at least about 96% sequence identity to the amino acid sequence of SEQ ID NO: 245. In some aspects, the pMHC complex comprises an amino acid sequence having at least about 97% sequence identity to the amino acid sequence of SEQ ID NO: 245. In some aspects, the pMHC complex comprises an amino acid sequence having at least about 98% sequence identity to the amino acid sequence of SEQ ID NO: 245. In some aspects, the pMHC complex comprises an amino acid sequence having at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 245. In some aspects, the pMHC complex comprises an amino acid sequence having 100% sequence identity to the amino acid sequence of SEQ ID NO: 245. In certain aspects of each of the foregoing embodiments, the peptide in the pMHC complex is identical to the peptide component of SEQ ID NO: 245 or has at most one or two amino acid substitutions.

[0188] In some embodiments, the pMHC complex comprises an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 247. In some embodiments, the pMHC complex comprises an amino acid sequence having at least about 91% sequence identity to the amino acid sequence of SEQ ID NO: 247. In some embodiments, the pMHC complex comprises an amino acid sequence having at least about 92% sequence identity to the amino acid sequence of SEQ ID NO: 247. In some embodiments, the pMHC complex comprises an amino acid sequence having at least about 93% sequence identity to the amino acid sequence of SEQ ID NO: 247. In some embodiments, the pMHC complex comprises an amino acid sequence having at least about 94% sequence identity to the amino acid sequence of SEQ ID NO: 247. In some embodiments, the pMHC complex comprises an amino acid sequence having at least about 95% sequence identity to the amino acid sequence of SEQ ID NO: 247. In some embodiments, the pMHC complex comprises an amino acid sequence having at least about 96% sequence identity to the amino acid sequence of SEQ ID NO: 247. In some aspects, the pMHC complex comprises an amino acid sequence having at least about 97% sequence identity to the amino acid sequence of SEQ ID NO: 247. In some aspects, the pMHC complex comprises an amino acid sequence having at least about 98% sequence identity to the amino acid sequence of SEQ ID NO: 247. In some aspects, the pMHC complex comprises an amino acid sequence having at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 247. In some aspects, the pMHC complex comprises an amino acid sequence having 100% sequence identity to the amino acid sequence of SEQ ID NO: 247. In certain aspects of each of the foregoing embodiments, the peptide in the pMHC complex is identical to the peptide component of SEQ ID NO: 247 or has at most one or two amino acid substitutions.

[0189] 6.4. Immune cell antigen targeting moieties Aspects of the present disclosure relate to molecules (e.g., T cell activators) that include immune cell antigen targeting moieties, i.e., targeting moieties that specifically bind to antigens present on or expressed by immune cells. Immune cell antigen targeting moieties generally bind to specific immune cell antigens, and in particular, to epitopes of immune cell antigens present on the surface of cells (e.g., extracellular domains or transmembrane proteins on the cell surface). Immune cell antigen targeting moieties of the present disclosure may be of a specific "type," i.e., may specifically bind to antigens expressed by specific types of immune cells (e.g., T cells, B cells, dendritic cells, etc.). T cell activators of the present disclosure may include one, two, three, or more immune cell antigen targeting moieties. When a T cell activator includes multiple immune cell antigen targeting moieties, each may be of the same type or different types. Types of immune cell antigen targeting moieties contemplated herein include, but are not limited to, T cell antigen targeting moieties and B cell antigen targeting moieties.

[0190] 6.4.1.T cell antigen targeting moiety Certain T cell activators of the present disclosure comprise one or more T cell antigen (TCA) targeting moieties. Without being bound by theory, it is understood that the incorporation of a T cell antigen (TCA) targeting moiety in a multispecific molecule of the present disclosure, in some embodiments, allows for activation of T cells even in the absence of antigen-presenting cells. For example, a T cell activator of the present disclosure may bind to a T cell receptor on a T cell via the pMHC complex and to a T cell antigen (e.g., CD3, TCR, CD28) on the T cell via the TCA targeting moiety, thereby activating the T cell in a manner specific for the particular antigen contained in the pMHC complex.

[0191] TCA targeting moieties of the present disclosure include targeting moieties (e.g., antibodies and antigen-binding fragments thereof) that bind to TCAs (e.g., CD3, CD28) with high affinity. TCA targeting moieties of the present disclosure also include targeting moieties (e.g., antibodies and antigen-binding fragments thereof) that bind to TCAs (e.g., CD3, CD28) with moderate or low affinity, depending on the therapeutic situation and the particular targeting properties desired. In some embodiments, "low affinity" (also "weak affinity") refers to a targeting moiety that binds to a TCA with less than 10 -6 Super M, 10 -7 Super M, 10 -8 Over M or 10 -9 K over M D or EC 50 represents a TCA targeting moiety that binds at (e.g., as measured by a surface plasmon resonance assay).

[0192] The present disclosure also includes TCA targeting moieties that bind to TCAs without measurable affinity. For example, in the context of a T cell activator comprising a pMHC complex and a CD3 targeting moiety, it may be desirable for the CD3 targeting moiety to bind to CD3 with only moderate or low affinity, or to bind to CD3 without measurable affinity. In this manner, preferential binding of pMHC to the TCR on T cells can be achieved while avoiding overall / non-targeted CD3 binding. Thus, in some embodiments, the TCA targeting moiety of the present disclosure has a binding affinity of 10 or more, as measured by surface plasmon resonance assay. -6 Super M, 10 -7 Super M, 10 -8 Over M or 10 -9 K over M D and any derivable range or value therein (e.g., 10 -6 M~10 -7 M, 10 -6 M~10 -8 M, 10 -6 M~10 -9 M, 10 -7 M~10 -8 M, or 10 -7 M~10 -9 M, 10 -8 M~10 -8M). Such TCA targeting moieties include those that have no detectable binding as measured by surface plasmon resonance assays and have a specific K D TCA targeting moieties described as "super" include TCA targeting moieties that have no detectable binding.

[0193] TCA targeting moieties generally bind to specific T cell antigens. Exemplary target molecules recognized by the TCA targeting moieties of the present disclosure are described in Section 6.4.1. Suitable targeting moiety formats are described in Section 6.4.3. The targeting moiety is preferably an antigen-binding portion, e.g., an antibody or an antigen-binding portion of an antibody, e.g., an scFv, as described in Section 6.4.3.1, or a Fab, as described in Section 6.4.3.2.

[0194] 6.4.1.1.T cell antigens Generally, the target of a TCA targeting moiety is any molecule present on or expressed by a T cell. In certain embodiments, the TCA targeting moiety of the present disclosure targets a cell surface molecule of a T cell. Exemplary targets of the TCA targeting moiety of the present disclosure include, but are not limited to, CD3, a T cell receptor (e.g., TCRαβ or TCRγδ), CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, and B7-H3. In some embodiments, the target of a TCA targeting moiety is CD3. In some embodiments, the target of a TCA targeting moiety is a T cell receptor (e.g., TCRαβ or TCRγδ). In some embodiments, the target of a TCA targeting moiety is CD28. The epitope of the TCA targeting moiety can be an individual polypeptide (eg, CD3 epsilon) or a multimeric component of a protein complex (eg, the TCRαβ or TCRγδ dimer of the T cell receptor complex).

[0195] CD3 and TCR Targeting Moieties In certain embodiments, the TCA targeting moiety of the present disclosure is a CD3 targeting moiety and / or a TCR targeting moiety. The CD3 targeting moiety may be or may include an antigen binding domain derived from an anti-CD3 antibody. The TCR targeting moiety may be or may include an antigen binding domain derived from an anti-TCR antibody.

[0196] Exemplary anti-CD3 and anti-TCR antibodies or antibody sequences upon which the TCA targeting moiety can be based are shown in Table G-1 below.

[0197] [Table 7-1]

[0198] [Table 7-2]

[0199] [Table 7-3]

[0200] [Table 7-4]

[0201] [Table 7-5]

[0202] [Table 7-6]

[0203] [Table 7-7]

[0204] In some aspects, the TCA targeting moiety competes with an antibody listed in Table G-1 for binding to a target (e.g., CD3 or a T cell receptor). In further aspects, the TCA targeting moiety comprises a CDR having the CDR sequence of an antibody listed in Table G-1. In some embodiments, the TCA targeting moiety comprises all six CDR sequences of an antibody listed in Table G-1. In other embodiments, the TCA targeting moiety comprises at least the heavy chain CDR sequences (CDR-H1, CDR-H2, CDR-H3) or antibody listed in Table G-1 and the light chain CDR sequence of a universal light chain. In further aspects, the TCA targeting moiety comprises a VH comprising the amino acid sequence of the VH of an antibody listed in Table G-1. In some embodiments, the TCA targeting moiety further comprises a VL comprising the amino acid sequence of the VL of an antibody listed in Table G-1. In other embodiments, the TCA targeting moiety further comprises a universal light chain VL sequence.

[0205] CD3 targeting moieties of the present disclosure include targeting moieties (e.g., antibodies and antigen-binding fragments thereof) that bind to CD3 (e.g., human CD3) with high affinity. CD3 targeting moieties of the present disclosure also include targeting moieties (e.g., antibodies and antigen-binding fragments thereof) that bind to CD3 (e.g., human CD3) with moderate or low affinity, depending on the therapeutic context and the particular targeting properties desired. The present disclosure also includes CD3 targeting moieties that bind to CD3 (e.g., human CD3) without measurable affinity. Thus, in some embodiments, CD3 targeting moieties of the present disclosure bind to CD3 (e.g., human CD3) with a binding affinity of 10 or less, as measured by surface plasmon resonance assay (e.g., at 25°C). -6 Super M, 10 -7 Super M, 10 -8 Over M or 10 -9 K over M D and any derivable range or value therein (e.g., 10 -6 M~10 -7 M, 10 -6 M~10 -8 M, 10 -6 M~10 -9 M, 10 -7 M~10 -8 M, or 10 -7 M~10-9 M, or 10 -8 M~10 -8 Such CD3 targeting moieties include those that have no detectable binding as measured by surface plasmon resonance assays and have a specific K D CD3 targeting moieties described as "super" include CD3 targeting moieties that do not have detectable binding. For example, in the context of a T cell activator comprising a CD3 targeting moiety and a pMHC complex, it may be desirable for the CD3 targeting moiety to bind to CD3 with moderate or low affinity, as thereby, as disclosed herein, T cell activators having CD3 binding moieties with moderate to weak affinity provide superior antigen-specific T cell activation compared to those having CD3 binding moieties with strong affinity (see, e.g., Examples 1-4 herein). In other embodiments, the CD3 targeting moieties of the present disclosure exhibit a binding affinity of 10 or more, as measured by surface plasmon resonance assay (e.g., at 25°C). -9 Under M, 10 -10 Under M, 10 -11 Less than M or 10 -12 K less than M D and any derivable range or value therein (e.g., 10 -9 M~10 -12 M, 10 -10 M~10 -12 M, 10 -11 M~10 -12 M, 10 -9 M~10 -10 M, 10 -9 M~10 -11 M, or 10 -10 M~10 -11 Includes M.

[0206] 6.4.1.1.2.CD28 targeting moiety In certain embodiments, the TCA targeting moiety of the present disclosure is a CD28 targeting moiety. The CD28 targeting moiety may be or may include the antigen-binding domain from an anti-CD28 antibody.

[0207] Exemplary anti-CD28 antibodies or antibody sequences upon which the TCA targeting moiety can be based are shown in Table G-2 below.

[0208] [Table 8]

[0209] In some aspects, the TCA targeting moiety competes with an antibody listed in Table G-2 for binding to a target (e.g., CD28). In further aspects, the TCA targeting moiety comprises a CDR having the CDR sequence of an antibody listed in Table G-2. In some embodiments, the TCA targeting moiety comprises all six CDR sequences of an antibody listed in Table G-2. In other embodiments, the TCA targeting moiety comprises at least the heavy chain CDR sequences (CDR-H1, CDR-H2, CDR-H3) or antibody listed in Table G-2 and the light chain CDR sequence of a universal light chain. In further aspects, the TCA targeting moiety comprises a VH comprising the amino acid sequence of the VH of an antibody listed in Table G-2. In some embodiments, the TCA targeting moiety further comprises a VL comprising the amino acid sequence of the VL of an antibody listed in Table G-2. In other embodiments, the TCA targeting moiety further comprises a universal light chain VL sequence.

[0210] CD28 targeting moieties of the present disclosure include targeting moieties (e.g., antibodies and antigen-binding fragments thereof) that bind to CD28 (e.g., human CD28) with high affinity. CD28 targeting moieties of the present disclosure also include targeting moieties (e.g., antibodies and antigen-binding fragments thereof) that bind to CD28 (e.g., human CD28) with moderate or low affinity, depending on the therapeutic context and the particular targeting properties desired. The present disclosure also includes CD28 targeting moieties that bind to CD28 (e.g., human CD28) without measurable affinity. Thus, in some embodiments, CD28 targeting moieties of the present disclosure bind to CD28 (e.g., human CD28) with a binding affinity of 10 or less, as measured by surface plasmon resonance assay. -6 Super M, 10 -7 Super M, 10 -8 Over M or 10 -9 K over M Dand any derivable range or value therein (e.g., 10 -6 M~10 -7 M, 10 -6 M~10 -8 M, 10 -6 M~10 -9 M, 10 -7 M~10 -8 M, or 10 -7 M~10 -9 M, 10 -8 M~10 -8 Such CD28 targeting moieties include those that have no detectable binding as measured by surface plasmon resonance assays and have a specific K D CD28 targeting moieties described as "super" include CD28 targeting moieties that do not have detectable binding. For example, in the context of a T cell activator comprising a CD28 targeting moiety and a pMHC complex, it may be desirable for the CD28 targeting moiety to bind to CD28 with only moderate or low affinity. In this manner, preferential targeting of the T cell activator to T cells expressing a TCR specific for the pMHC complex can be achieved while avoiding global / non-targeted CD28 binding and the resulting adverse side effects associated therewith.

[0211] In other embodiments, the CD28 targeting moiety of the present disclosure has a binding activity of 10 or more, as measured by a surface plasmon resonance assay (e.g., at 25°C). -9 Under M, 10 -10 Under M, 10 -11 Less than M or 10 -12 K less than M D and any derivable range or value therein (e.g., 10 -9 M~10 -12 M, 10 -10 M~10 -12 M, 10 -11 M~10 -12 M, 10 -9 M~10 -10 M, 10 -9 M~10 -11 M, or 10 -10 M~10 -11 Includes M.

[0212] 6.4.2.B cell antigen targeting moiety Certain T cell activators of the present disclosure comprise one or more B cell antigen (BCA) targeting moieties. BCA targeting moieties generally bind to specific B cell antigens. Exemplary target molecules recognized by targeting moieties of the present disclosure are described in Section 6.4.2.1.

[0213] Suitable targeting moiety formats are described in Section 6.4.3. The targeting moiety is preferably an antigen-binding portion, e.g., an antibody or an antigen-binding portion of an antibody, e.g., an scFv, as described in Section 6.4.3.1, or a Fab, as described in Section 6.4.3.2.

[0214] In some embodiments, a T cell activator of the present disclosure that includes a BCA targeting moiety lacks a TCA (e.g., CD3) targeting moiety. In other embodiments, a T cell activator of the present disclosure that includes a BCA targeting moiety also includes a TCA (e.g., CD3) targeting moiety.

[0215] 6.4.2.1.B cell antigens Generally, the target of a BCA targeting moiety is any molecule present on or expressed by B cells. In certain embodiments, the BCA targeting moiety of the present disclosure targets a cell surface molecule of a B cell. Exemplary targets of the BCA targeting moiety of the present disclosure include, but are not limited to, CD19, CD20, and CD22. In some embodiments, the target of a BCA targeting moiety is CD19. In some embodiments, the target of a BCA targeting moiety is CD20. In some embodiments, the target of a BCA targeting moiety is CD22. The epitope of a BCA targeting moiety can be an individual polypeptide or a multimeric component of a protein complex.

[0216] 6.4.2.1.1.CD19 targeting moiety In certain embodiments, a BCA targeting moiety of the present disclosure is a CD19 targeting moiety. The CD19 targeting moiety may be or may include an antigen-binding domain derived from an anti-CD19 antibody.

[0217] Exemplary anti-CD19 antibodies or antibody sequences upon which a BCA targeting moiety can be based are shown in Table G-3 below.

[0218] [Table 9]

[0219] In some aspects, the BCA targeting moiety competes with an antibody listed in Table G-3 for binding to a target. In further aspects, the BCA targeting moiety comprises a CDR having the CDR sequence of an antibody listed in Table G-3. In some embodiments, the BCA targeting moiety comprises all six CDR sequences of an antibody listed in Table G-3. In other embodiments, the BCA targeting moiety comprises at least the heavy chain CDR sequences (CDR-H1, CDR-H2, CDR-H3) or antibody listed in Table G-3 and the light chain CDR sequence of a universal light chain. In further aspects, the BCA targeting moiety comprises a VH comprising the amino acid sequence of the VH of an antibody listed in Table G-3. In some embodiments, the BCA targeting moiety further comprises a VL comprising the amino acid sequence of the VL of an antibody listed in Table G-3. In other embodiments, the BCA targeting moiety further comprises a universal light chain VL sequence.

[0220] 6.4.2.1.2.CD20 targeting moiety In certain embodiments, a BCA targeting moiety of the present disclosure is a CD20 targeting moiety. The CD20 targeting moiety may be or may include an antigen-binding domain derived from an anti-CD20 antibody.

[0221] Exemplary anti-CD20 antibodies or antibody sequences upon which a BCA targeting moiety can be based are shown in Table G-4 below.

[0222] [Table 10-1]

[0223] [Table 10-2]

[0224] In some aspects, the BCA targeting moiety competes with an antibody listed in Table G-4 for binding to a target. In further aspects, the BCA targeting moiety comprises a CDR having the CDR sequence of an antibody listed in Table G-4. In some embodiments, the BCA targeting moiety comprises all six CDR sequences of an antibody listed in Table G-4. In other embodiments, the BCA targeting moiety comprises at least the heavy chain CDR sequences (CDR-H1, CDR-H2, CDR-H3) or antibody listed in Table G-4 and the light chain CDR sequence of a universal light chain. In further aspects, the BCA targeting moiety comprises a VH comprising the amino acid sequence of the VH of an antibody listed in Table G-4. In some embodiments, the BCA targeting moiety further comprises a VL comprising the amino acid sequence of the VL of an antibody listed in Table G-4. In other embodiments, the BCA targeting moiety further comprises a universal light chain VL sequence.

[0225] 6.4.2.1.3.CD22 targeting moiety In certain embodiments, a BCA targeting moiety of the present disclosure is a CD22 targeting moiety. The CD22 targeting moiety may be or may include an antigen-binding domain derived from an anti-CD22 antibody.

[0226] Exemplary anti-CD22 antibodies or antibody sequences upon which a BCA targeting moiety can be based are shown in Table G-5 below.

[0227] [Table 11]

[0228] In some aspects, the BCA targeting moiety competes with an antibody listed in Table G-5 for binding to a target. In further aspects, the BCA targeting moiety comprises a CDR having the CDR sequence of an antibody listed in Table G-5. In some embodiments, the BCA targeting moiety comprises all six CDR sequences of an antibody listed in Table G-5. In other embodiments, the BCA targeting moiety comprises at least the heavy chain CDR sequences (CDR-H1, CDR-H2, CDR-H3) or antibody listed in Table G-5 and the light chain CDR sequence of a universal light chain. In further aspects, the BCA targeting moiety comprises a VH comprising the amino acid sequence of the VH of an antibody listed in Table G-5. In some embodiments, the BCA targeting moiety further comprises a VL comprising the amino acid sequence of the VL of an antibody listed in Table G-5. In other embodiments, the BCA targeting moiety further comprises a universal light chain VL sequence.

[0229] 6.4.3. Targeting Part Format In certain aspects, the targeting moiety can be any type of antibody or fragment thereof that retains specific binding to an antigenic determinant. In one embodiment, the antigen-binding moiety is a full-length antibody. In one embodiment, the antigen-binding moiety is an immunoglobulin molecule, particularly an immunoglobulin molecule of the IgG class, more particularly an IgG1 or IgG4 immunoglobulin molecule. Antibody fragments include VH (or V H ) fragment, VL (or V L ) fragments, Fab fragments, F(ab')2 fragments, scFv fragments, Fv fragments, VHH fragments, minibodies, intrabodies, diabodies, triabodies, and tetrabodies.

[0230] 6.4.3.1.scFv Single-chain Fv or "scFv" antibody fragments comprise the VH and VL domains of an antibody within a single polypeptide chain, can be expressed as single-chain polypeptides, and retain the specificity of the intact antibody from which they are derived. Generally, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for target binding. Examples of linkers suitable for linking the VH and VL chains of an scFv are the linkers identified in Section 6.8.

[0231] As used herein, unless otherwise specified, an scFv may have a VL variable region and a VH variable region in either order, e.g., with respect to the N-terminus and C-terminus of the polypeptide, and may comprise a VL-linker-VH or a VH-linker-VL.

[0232] The scFv can comprise VH and VL sequences from any suitable species, such as murine, human, or humanized VH and VL sequences. To generate nucleic acids encoding scFvs, DNA fragments encoding the VH and VL are operably linked to another fragment encoding a linker, for example, a fragment encoding any of the linkers described in Section 6.8 (typically a repeat of a sequence containing the amino acids glycine and serine, such as the amino acid sequence (Gly4-Ser)3 (SEQ ID NO: 127)), such that the VH and VL sequences can be expressed as a contiguous single-chain protein with the VL and VH regions connected by a flexible linker (see, e.g., Bird et al., 1988, Science 242:423-426; Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; McCafferty et al., 1990, Nature 348:552-554).

[0233] 6.4.3.2.Fab Fab domains are traditionally generated by proteolytic cleavage of immunoglobulin molecules using enzymes such as papain. In the T cell activators of the present disclosure, the Fab domain is typically recombinantly expressed as part of the T cell activator.

[0234] The Fab domain can comprise constant domain and variable region sequences from any suitable species, and thus can be murine, chimeric, human, or humanized.

[0235] A Fab domain typically comprises a CH1 domain attached to a VH domain, which pairs with a CL domain attached to a VL domain. In wild-type immunoglobulins, the VH domain pairs with the VL domain to form the Fv region, and the CH1 domain pairs with the CL domain to further stabilize the link module. Disulfide bonds between the two constant domains can further stabilize the Fab domain.

[0236] For the molecules of the present disclosure, particularly when the light chain is not a common or universal light chain, it is advantageous to use a Fab heterodimerization strategy to allow correct association of Fab domains belonging to the same ABD and minimize aberrant pairing of Fab domains belonging to different ABDs. For example, the Fab heterodimerization strategy shown in Table 3 below can be used:

[0237] [Table 12]

[0238] Thus, in certain embodiments, correct association between the two polypeptides of a Fab is facilitated by exchanging the VL and VH domains of the Fab with one another, or by exchanging the CH1 and CL domains with one another, as described, for example, in WO2009 / 080251.

[0239] Correct Fab pairing can also be promoted by introducing one or more amino acid modifications in the CH1 domain and one or more amino acid modifications in the CL domain of the Fab, and / or by introducing one or more amino acid modifications in the VH domain and one or more amino acid modifications in the VL domain of the Fab. The modified amino acids are typically part of the VH:VL and CH1:CL interfaces such that the Fab components preferentially pair with each other rather than with other Fab components.

[0240] In one embodiment, the one or more amino acid modifications are limited to conserved framework residues of the variable (VH, VL) and constant (CH1, CL) domains, as indicated by the Kabat numbering of the residues. Almagro, 2008, Frontiers In Bioscience 13:1619-1633 provides definitions of framework residues based on the Kabat, Chothia, and IMGT numbering schemes.

[0241] In one embodiment, the modifications introduced in the VH and CH1 and / or VL and CL domains are complementary to each other. Complementarity at the heavy and light chain interface can be achieved based on steric and hydrophobic contacts, electrostatic / charge interactions, or a combination of various interactions. Complementarity between protein surfaces has been widely described in the literature in terms of lock and key fit, knob into hole, protrusion and cavity, donor and acceptor, etc., all of which suggest the nature of the structural and chemical match between the two interacting surfaces.

[0242] In one embodiment, one or more of the introduced modifications introduce new hydrogen bonds across the interface of the Fab component. In one embodiment, one or more of the introduced modifications introduce new salt bridges across the interface of the Fab component. Exemplary substitutions are described in WO2014 / 150973 and WO2014 / 082179, the contents of which are incorporated herein by reference.

[0243] In some embodiments, the Fab domain comprises a 192E substitution in the CH1 domain and 114A and 137K substitutions in the CL domain, which introduces a salt bridge between the CH1 and CL domains (see, e.g., Golay et al., 2016, J Immunol 196:3199-211).

[0244] In some embodiments, the Fab domain comprises 143Q and 188V substitutions in the CH1 domain and 113T and 176V substitutions in the CL domain, which serve to exchange hydrophobic and polar contact regions between the CH1 and CL domains (see, e.g., Golay et al., 2016, J Immunol 196:3199-211).

[0245] In some embodiments, the Fab domain can include modifications in some or all of the VH, CH1, VL, and CL domains to introduce an orthogonal Fab interface that promotes correct assembly of the Fab domain (Lewis et al., 2014 Nature Biotechnology 32:191-198). In embodiments, a 39K, 62E modification is introduced in the VH domain, an H172A, F174G modification is introduced in the CH1 domain, a 1R, 38D, (36F) modification is introduced in the VL domain, and an L135Y, S176W modification is introduced in the CL domain. In another embodiment, a 39Y modification is introduced in the VH domain and a 38R modification is introduced in the VL domain.

[0246] Fab domains can also be modified to replace the native CH1:CL disulfide bond with an engineered disulfide bond, thereby increasing the efficiency of pairing of the Fab components. For example, an engineered disulfide bond can be introduced by introducing 126C into the CH1 domain and 121C into the CL domain (see, e.g., Mazor et al., 2015, MAbs 7:377-89).

[0247] Fab domains can also be modified by replacing the CH1 and CL domains with alternative domains that promote correct assembly. For example, Wu et al., 2015, MAbs 7:364-76, describe replacing the CH1 domain with a constant domain of a T cell receptor and the CL domain with a b domain of a T cell receptor, pairing these domain replacements with additional charge-charge interactions between the VL and VH domains by introducing a 38D modification in the VL domain and a 39K modification in the VH domain.

[0248] Alternatively, or in addition to using a Fab heterodimerization strategy to promote correct VH-VL pairing, a VL of a common light chain (also referred to as a universal light chain) can be used in each Fab VL region of the T cell activator of the present disclosure. In various embodiments, using a common light chain as described herein reduces the number of incorrect species of the T cell activator compared to using the original cognate VL. In various embodiments, the VL domain of the T cell activator is identified from a monospecific antibody that comprises a common light chain. In various embodiments, the VH region of the T cell activator comprises human heavy chain variable gene segments that are rearranged in vivo in mouse B cells that have been previously engineered to express a limited human light chain repertoire or a single human light chain, cognate to the human heavy chain, and in response to exposure to an antigen of interest, generates an antibody repertoire that includes one of two possible human VLs or multiple human VHs that are cognate to one of the human heavy chains, and this antibody repertoire is specific for the antigen of interest. The common light chain is derived from a rearranged human Vκ1-39Jκ5 sequence or a rearranged human Vκ3-20Jκ1 sequence, including somatically mutated (e.g., affinity matured) forms. See, e.g., U.S. Patent No. 10,412,940.

[0249] 6.5. Tumor antigen targeting moiety The T cell activators of the present disclosure may optionally include at least one targeting moiety that specifically binds to a target molecule expressed by tumor cells or within the tumor cell environment, such as an extracellular matrix ("ECM") antigen, a tumor-reactive lymphocyte antigen, a cell surface molecule of tumor or viral lymphocytes, a checkpoint inhibitor, or a tumor-associated antigen (TAA), collectively referred to herein as a "tumor antigen targeting moiety." Those skilled in the art will recognize that the aforementioned categories of target molecules are not mutually exclusive, and thus, a given target molecule may be classified into more than one of the aforementioned categories of target molecules. For example, some molecules may be considered both a TAA and an ECM protein. Preferably, the ECM antigen, tumor-reactive lymphocyte antigen, cell surface molecule of tumor or viral lymphocytes, a checkpoint inhibitor, or a TAA is a human antigen. The antigen may or may not be present on normal cells. Certain embodiments are directed to T cell activators that include at least one targeting moiety that specifically binds to a TAA.

[0250] It is expected that any type of tumor and any type of ECM antigen, tumor-reactive lymphocyte antigen, cell surface molecule of tumor or viral lymphocyte, checkpoint inhibitor, or TAA can be targeted by the T cell activators of the present disclosure. Exemplary types of cancer that can be targeted include acute lymphoblastic leukemia, acute myeloid leukemia, biliary tract cancer, B-cell leukemia, B-cell lymphoma, biliary tract cancer, bone cancer, brain cancer, breast cancer, triple-negative breast cancer, cervical cancer, Burkitt's lymphoma, chronic lymphocytic leukemia, chronic myeloid leukemia, colorectal cancer, endometrial cancer, esophageal cancer, gallbladder cancer, gastric cancer, gastrointestinal tract cancer, glioma, hairy cell leukemia, head and neck cancer, Hodgkin's lymphoma, liver cancer, lung cancer, medullary thyroid carcinoma, melanoma, multiple myeloma, ovarian cancer, non-Hodgkin's lymphoma, pancreatic cancer, prostate cancer, lung duct cancer, renal cancer, sarcoma, skin cancer, testicular cancer, urothelial carcinoma and other bladder cancer.However, those skilled in the art will recognize that TAAs and other target molecules related to tumor microenvironment are known for virtually any type of cancer.

[0251] Non-limiting examples of ECM antigens include syndecans, heparanase, integrins, osteopontin, link, cadherins, laminins, laminin-type EGFs, lectins, fibronectin, notch, nectins (e.g., nectin-4), tenascins, collagens (e.g., type X collagen), and matrixins.

[0252] In certain embodiments, the target molecule is a checkpoint inhibitor, e.g., CTLA-4, PD1, PDL1, PDL2, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK1, or CHK2. In certain embodiments, the target molecule is PD1. In other embodiments, the target molecule is LAG3. In some embodiments where the target molecule is a checkpoint inhibitor, the tumor antigen targeting moiety does not block or only poorly blocks ligand-receptor binding. Examples of non-blocking or low-blocking anti-PD1 antibodies include antibodies having the VH / VL amino acid sequences of SEQ ID NOs: 2 / 10 in WO 2015 / 112800 A1, SEQ ID NOs: 16 / 17 in U.S. Pat. No. 11,034,765 B2, and SEQ ID NOs: 164 / 178, 165 / 179, 166 / 180, 167 / 181, 168 / 182, 169 / 183, 170 / 184, 171 / 185, 172 / 186, 173 / 187, 174 / 188, 175 / 189, 176 / 190, and 177 / 190 in U.S. Pat. No. 10,294,299 B2. Examples of non-blocking or low-blocking anti-LAG3 antibodies include antibodies having the VH / VL amino acid sequences of SEQ ID NOs: 23 / 24, 3 / 4, and 11 / 12 of US Patent Application Publication No. 2022 / 0056126A1.

[0253] In certain embodiments, the target molecule of the tumor antigen targeting moiety is a TAA. Exemplary TAAs are set forth in Table H below, along with reference to exemplary antibodies or antibody sequences on which the tumor antigen targeting moiety can be based.

[0254] [Table 13-1]

[0255] Table 13-2

[0256] Table 13-3

[0257] Table 13-4

[0258] Table 13-5

[0259] Table 13-6

[0260] Table 13-7

[0261] Table 13-8

[0262] Table 13-9

[0263] Table 13-10

[0264] Table 13-11

[0265] Table 13-12

[0266] Table 13-13

[0267] Table 13-14

[0268] Table 13-15

[0269] Table 13-16

[0270] Table 13-17

[0271] Table 13-18

[0272] Table 13-19

[0273] Table 13-20

[0274] In some aspects, the tumor antigen targeting moiety competes with an antibody set forth in Table H for binding to a TAA. In further aspects, the tumor antigen targeting moiety comprises a CDR having the CDR sequence of an anti-TAA antibody set forth in Table H. In some embodiments, the tumor antigen targeting moiety comprises all six CDR sequences of an anti-TAA antibody set forth in Table H. In other embodiments, the tumor antigen targeting moiety comprises at least the heavy chain CDR sequences (CDR-H1, CDR-H2, CDR-H3) and the light chain CDR sequences of a universal light chain. In a further aspect, the tumor antigen targeting moiety comprises a VH comprising the amino acid sequence of the VH of an anti-TAA antibody listed in Table H. In some embodiments, the tumor antigen targeting moiety further comprises a VL comprising the amino acid sequence of the VL of an anti-TAA antibody listed in Table H. In other embodiments, the tumor antigen targeting moiety further comprises a universal light chain VL sequence. Additional TAAs that can be targeted by the tumor antigen targeting moiety are disclosed, for example, in Hafeez et al., 2020, Molecules 25:4764, doi:10.3390 / molecules25204764, particularly Table 1. Table 1 of Hafeez et al. is incorporated herein by reference in its entirety.

[0275] Further exemplary TAAs include fibroblast activation protein (FAP), the A1 domain of tenascin-C (TNC A1), the A2 domain of tenascin-C (TNC A2), fibronectin extra domain B (EDB), melanoma-associated chondroitin sulfate proteoglycan (MCSP), MART-1 / Melan-A, gp100, dipeptidyl peptidase IV (DPPIV), adenosine deaminase-binding protein (ADAbp), cyclophilin b, colorectal-associated antigen (CRC)-C017-1A / GA733, carcinoembryonic antigen (CEA) and its immunogenic epitopes CAP-1 and CAP-2, etv6, aml1, prostate-specific antigen (PSA) and its immunogenic epitopes PSA-1, PSA-2, and PSA-3, prostate-specific membrane antigen (PSMA), T-cell receptor / CD3-zeta chain, MAGE-tumor antigen family (e.g., MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAG MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A12, MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE-Xp4 (MAGE-B4), MAGE-C1, MAGE-C2, MAGE-C3, MAGE-C4, MAGE-C5), GAGE-tumor antigen family (e.g., GAGE-1, GAGE-2, GAGE- 3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8, GAGE-9), BAGE, RAGE, LAGE-1, NAG, GnT-V, MUM-1, CDK4, tyrosinase, p53, MUC family, HER2 / neu, p21ras, RCAS1, α-fetoprotein, E-cadherin, α-catenin, β-catenin, and γ-catenin, p120ctn, gp100Pmel117, PRAME, NY-ESO-1, cdc27, adenomatous polyposis coli protein (APC), fodrin, connexin 37, Ig-idiotypes, p15, gp75, GM2, and GD2 gangliosides, viral products such as human papillomavirus proteins, the Smad family of tumor antigens, Imp-1, P1A, EBV-encoded nuclear antigen (EBNA)-1, brain glycogen phosphorylase, SSX-1, SSX-2 (HOM-MEL-40), SSX-1, SSX-4, SSX-5, SCP-1, and CT-7, c-erbB-2, Her2, EGFR, IGF-1R, CD2 (T cell surface antigen), CD3 (T cell receptor-related antigen), and CD22 (B cell receptor), CD23 (low affinity IgE receptor), CD30 (cytokine receptor), CD33 (myeloid cell surface antigen), CD40 (tumor necrosis factor receptor), IL-6R (IL6 receptor), CD20, MCSP, PDGFβR (β-platelet-derived growth factor receptor), ErbB2 epithelial cell adhesion molecule (EpCAM), EGFR variant III (EGFRvIII), CD19, disialoganglioside GD2, ductal epithelial mucin, gp36, TAG-72, glioma-associated antigen, β-human chorionic gonadotropin, alpha-fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxylesterase, mut hsp70-2, M-CSF, prostase, prostate-specific antigen (PSA), PAP, LAGA-1a, p53, prostein, PSMA, survival and telomerase, prostate cancer tumor antigen-1 (PCTA-1), ELF2M, neutrophil elastase, ephrin B2, insulin growth factor (IGF1)-I, IGF-II, IGF1 receptor, 5T4, ROR1, Nkp30, NKG2D, tumor stromal antigen, extra domain A (EDA) and extra domain B (EDB) of fibronectin, and the A1 domain of tenascin-C (TnC A1).

[0276] 6.6. Multispecific antigen binding molecules Aspects of the present disclosure are directed to compositions comprising a T cell activator and a multispecific antigen-binding molecule, as well as methods comprising administering a T cell activator and a multispecific antigen-binding molecule (together or separately). As used herein, a multispecific antigen-binding molecule refers to a molecule comprising (a) at least one tumor antigen targeting moiety or at least one BCA targeting moiety, and (b) at least one TCA targeting moiety. A multispecific antigen-binding molecule may further comprise one or more additional tumor antigen targeting moieties, one or more additional BCA targeting moieties, and / or one or more additional TCA targeting moieties. Generally, a "multispecific antigen-binding molecule" as used herein refers to a molecule that does not comprise a pMHC complex. Pharmaceutical compositions comprising such multispecific antigen-binding molecules, in some cases together with a T cell activator disclosed herein, are also disclosed. In some embodiments, methods for using such multispecific antigen-binding molecules in the treatment of cancer in combination with a T cell activator disclosed herein are further disclosed.

[0277] Certain exemplary tumor-associated antigens and related indications are provided in Table K-2. In some embodiments, the multispecific antigen binding molecule comprises a tumor antigen targeting moiety that specifically binds to a TAA of Table K-1. In some embodiments, the TAA is BCMA. In some embodiments, the TAA is CD19. In some embodiments, the TAA is CD20. In some embodiments, the TAA is CD22. In some embodiments, the TAA is EGFR.

[0278] [Table 14-1]

[0279] [Table 14-2]

[0280] [Table 14-3]

[0281] In some embodiments, the multispecific antigen-binding molecule comprises a BCA targeting moiety that specifically binds to CD19, CD20, or CD22. In some embodiments, the BCA targeting moiety specifically binds to CD19. In some embodiments, the BCA targeting moiety specifically binds to CD20. In some embodiments, the BCA targeting moiety specifically binds to CD22.

[0282] Certain exemplary multispecific antigen-binding molecules are provided in Table K-2. In some embodiments, a multispecific antigen-binding molecule of the present disclosure is a bispecific antigen-binding molecule of Table K-2. In some embodiments, a multispecific antigen-binding molecule comprises one or more CDR, VH, and / or VL sequences from a bispecific antigen-binding molecule of Table K-2.

[0283] [Table 15-1]

[0284] [Table 15-2]

[0285] [Table 15-3]

[0286] 6.7. Multimerization moiety Embodiments of the present disclosure are directed to multispecific molecules (e.g., T cell activators) that comprise a multimerization moiety. A "multimerization moiety" refers to any polypeptide or other molecule or portion thereof that allows for multimerization (e.g., dimerization). Such multimerization includes, for example, the non-covalent association of two or more multimerization moieties. A variety of multimerization moieties are recognized in the art and are contemplated herein. Exemplary multimerization moieties of the present disclosure are further described below.

[0287] Fc Domain Multispecific molecules (e.g., T cell activators) of the present disclosure typically comprise a pair of Fc domains that associate to form an Fc region. In natural antibodies, Fc regions include a hinge region at their N-terminus to form a constant domain. Throughout this disclosure, reference to an Fc domain encompasses an Fc domain with or without a hinge domain. In some embodiments, an Fc domain includes a hinge domain at its N-terminus.

[0288] The Fc domain can be derived from any suitable species and operably linked to the pMHC complex, targeting moiety, or component thereof. In one embodiment, the Fc domain is derived from a human Fc domain. In a preferred embodiment, the pMHC complex, targeting moiety, or component thereof is fused to an IgG Fc molecule. The pMHC complex, targeting moiety, or component thereof can be fused to the N-terminus, C-terminus, or both of the IgG Fc domain.

[0289] The Fc domain can be derived from any suitable class of antibody, including IgA (including subclasses IgA1 and IgA2), IgD, IgE, IgG (including subclasses IgG1, IgG2, IgG3, and IgG4), and IgM. In one embodiment, the Fc domain is derived from IgG1, IgG2, IgG3, or IgG4. In one embodiment, the Fc domain is derived from IgG1. In one embodiment, the Fc domain is derived from IgG4.

[0290] The two Fc domains within an Fc region may be identical or different from one another. In natural antibodies, the Fc domains are typically identical, but for purposes of producing multispecific molecules, the Fc domains may advantageously be different to allow heterodimerization, as described in Section 6.7.1.2 below.

[0291] In natural antibodies, the heavy chain Fc domain of IgA, IgD, and IgG consists of two heavy chain constant domains (CH2 and CH3), while the domain of IgE and IgM consists of three heavy chain constant domains (CH2, CH3, and CH4), which dimerize to create the Fc region.

[0292] In the multispecific molecules of the disclosure, the Fc region, and / or Fc domains therein, can comprise heavy chain constant domains from antibodies of one or more different classes, e.g., 1, 2, or 3 different classes.

[0293] In one embodiment, the Fc region comprises the CH2 and CH3 domains derived from IgG1. In one embodiment, the Fc region comprises the CH2 and CH3 domains derived from IgG2.

[0294] In one embodiment, the Fc region comprises the CH2 and CH3 domains derived from IgG3. In one embodiment, the Fc region comprises the CH2 and CH3 domains derived from IgG4.

[0295] In one embodiment, the Fc region comprises a CH4 domain from IgM. The IgM CH4 domain is typically located C-terminal to the CH3 domain. In one embodiment, the Fc region comprises a CH2 domain and a CH3 domain derived from an IgG, and a CH4 domain derived from an IgM.

[0296] It will be understood that heavy chain constant domains for use in producing Fc regions for the multispecific molecules of the present disclosure can include variants of the naturally occurring constant domains described above. Such variants can include one or more amino acid mutations compared to the wild-type constant domain. In one example, the Fc region of the present disclosure includes at least one constant domain that differs in sequence from the wild-type constant domain. It will be understood that the variant constant domain can be longer or shorter than the wild-type constant domain. Preferably, the variant constant domain is at least 60% identical or similar to the wild-type constant domain. In another example, the variant constant domain is at least 70% identical or similar. In another example, the variant constant domain is at least 80% identical or similar. In another example, the variant constant domain is at least 90% identical or similar. In another example, the variant constant domain is at least 95% identical or similar.

[0297] IgM and IgA naturally occur in humans as covalently linked multimers of a common H2L2 antibody unit. IgM exists as a pentamer when a J chain is incorporated and as a hexamer when the J chain is absent. IgA exists in both monomeric and dimeric forms. The heavy chains of IgM and IgA have an 18-amino acid extension to the C-terminal constant domain known as the tail. The tail contains cysteine ​​residues that form disulfide bonds between heavy chains within the polymer and is thought to play an important role in polymerization. The tail also contains glycosylation sites. In certain embodiments, the multispecific molecules of the present disclosure do not contain a tail.

[0298] The Fc domains incorporated into the multispecific molecules of the present disclosure may contain one or more modifications that alter the functional properties of the protein, for example, binding to an Fc receptor such as FcRn or a leukocyte receptor, binding to complement, modified disulfide bond structures, or altered glycosylation patterns. Exemplary Fc modifications that alter effector function are described in Section 6.7.1.1.

[0299] Fc domains can also be altered to include modifications that improve the manufacturability of asymmetric multispecific molecules, for example, by enabling heterodimerization, the preferential pairing of non-identical Fc domains with identical Fc domains. Heterodimerization allows for the production of multispecific molecules in which different polypeptide components are connected to each other by Fc regions that contain Fc domains that differ in sequence. Examples of heterodimerization strategies are illustrated in Section 6.7.1.2.

[0300] It will be appreciated that any of the above modifications can be combined in any suitable way to achieve the desired functional properties and / or combined with other modifications to alter the properties of the multispecific molecule.

[0301] Exemplary Fc domain sequences are provided in Table F-1 below.

[0302] [Table 16-1]

[0303] [Table 16-2]

[0304] [Table 16-3]

[0305] In some embodiments, the Fc domain comprises an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to any one of the sequences disclosed in Table F-1.

[0306] In some embodiments, the Fc domain comprises an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO: 10. When an Fc domain comprises at least 90% sequence identity and less than 100% sequence identity to SEQ ID NO: 10 (e.g., 90%-99% sequence identity to SEQ ID NO: 10), the Fc domain may also comprise one or more amino acid substitutions described herein, e.g., one or more substitutions that reduce effector function (e.g., as described in Section 6.7.1.1) and / or one or more substitutions that promote Fc heterodimerization (e.g., as described in Section 6.7.1.2).

[0307] In some embodiments, the Fc domain comprises an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO: 11. When an Fc domain comprises at least 90% sequence identity and less than 100% sequence identity to SEQ ID NO: 11 (e.g., 90%-99% sequence identity to SEQ ID NO: 11), the Fc domain may also comprise one or more amino acid substitutions described herein, e.g., one or more substitutions that reduce effector function (e.g., as described in Section 6.7.1.1) and / or one or more substitutions that promote Fc heterodimerization (e.g., as described in Section 6.7.1.2).

[0308] In some embodiments, the Fc domain comprises an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO: 11. When an Fc domain comprises at least 90% sequence identity and less than 100% sequence identity to SEQ ID NO: 11 (e.g., 90%-99% sequence identity to SEQ ID NO: 11), the Fc domain may also comprise one or more amino acid substitutions described herein, e.g., one or more substitutions that reduce effector function (e.g., as described in Section 6.7.1.1) and / or one or more substitutions that promote Fc heterodimerization (e.g., as described in Section 6.7.1.2).

[0309] In some embodiments, the Fc domain comprises an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO: 12. When an Fc domain comprises at least 90% sequence identity and less than 100% sequence identity to SEQ ID NO: 12 (e.g., 90%-99% sequence identity to SEQ ID NO: 12), the Fc domain may also comprise one or more amino acid substitutions described herein, e.g., one or more substitutions that reduce effector function (e.g., as described in Section 6.7.1.1) and / or one or more substitutions that promote Fc heterodimerization (e.g., as described in Section 6.7.1.2).

[0310] In some embodiments, the Fc domain comprises an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO:13.

[0311] In some embodiments, the Fc domain comprises an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO:14.

[0312] In some embodiments, the Fc domain comprises an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO:15.

[0313] In some embodiments, the Fc domain comprises an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO:16.

[0314] In some embodiments, the Fc domain comprises an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO:17.

[0315] In some embodiments, the Fc domain comprises an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO:18.

[0316] In some embodiments, the Fc domain comprises an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO:19.

[0317] In some embodiments, the Fc domain comprises an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO:20.

[0318] In some embodiments, the Fc domain comprises an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO:21.

[0319] In some embodiments, the Fc domain comprises an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO:22.

[0320] In some embodiments, the Fc domain comprises an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO:23.

[0321] In some embodiments, the Fc domain comprises an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO:24.

[0322] In some embodiments, the Fc domain comprises an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO:25.

[0323] In some embodiments, the Fc domain comprises an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO:26.

[0324] In some embodiments, the Fc domain comprises an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO:27.

[0325] In some embodiments, the Fc domain comprises an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO:28.

[0326] 6.7.1.1. Fc Domains with Altered Effector Function In some embodiments, the Fc domain comprises one or more amino acid substitutions that reduce binding to Fc receptors and / or effector function.

[0327] In a specific embodiment, the Fc receptor is an Fcγ receptor. In one embodiment, the Fc receptor is a human Fc receptor. In one embodiment, the Fc receptor is an activating Fc receptor. In a specific embodiment, the Fc receptor is an activating human Fcγ receptor, more specifically, human FcγRIIIa, FcγRI, or FcγRIIa, most specifically, human FcγRIIIa. In one embodiment, the effector function is one or more selected from the group consisting of complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and cytokine secretion. In a specific embodiment, the effector function is ADCC.

[0328] In one embodiment, the Fc domain or Fc region (e.g., one or both Fc domains of a multispecific molecule that can associate to form an Fc region) comprises an amino acid substitution at a position selected from the group of E233, L234, L235, N297, P331, and P329 (numbering according to Kabat EU index). In a more particular embodiment, the Fc domain or Fc region comprises an amino acid substitution at a position selected from the group of L234, L235, and P329 (numbering according to Kabat EU index). In some embodiments, the Fc domain or Fc region comprises the amino acid substitutions L234A and L235A (numbering according to Kabat EU index). In one such embodiment, the Fc domain or region is an Igd Fc domain or region, particularly a human Igd Fc domain or region. In one embodiment, the Fc domain or Fc region comprises an amino acid substitution at position P329. In a more particular embodiment, the amino acid substitution is P329A or P329G, particularly P329G (numbering according to the Kabat EU index). In one embodiment, the Fc domain or Fc region comprises an amino acid substitution at position P329 and a further amino acid substitution at a position selected from E233, L234, L235, N297 and P331 (numbering according to the Kabat EU index). In a more particular embodiment, the further amino acid substitution is E233P, L234A, L235A, L235E, N297A, N297D or P331S. In a particular embodiment, the Fc domain or Fc region comprises amino acid substitutions at positions P329, L234 and L235 (numbering according to the Kabat EU index). In a more specific embodiment, the Fc domain comprises the amino acid mutations L234A, L235A and P329G (“P329G LALA,” “PGLALA,” or “LALAPG”).

[0329] Typically, the same one or more amino acid substitutions are present in each of the two Fc domains of the Fc region. Thus, in certain embodiments, each Fc domain of the Fc region comprises the amino acid substitutions L234A, L235A, and P329G (Kabat EU index numbering), i.e., in each of the first and second Fc domains of the Fc region, the leucine residue at position 234 is replaced with an alanine residue (L234A), the leucine residue at position 235 is replaced with an alanine residue (L235A), and the proline residue at position 329 is replaced with a glycine residue (P329G) (Kabat EU index numbering).

[0330] In one embodiment, the Fc domain is an IgG1 Fc domain, particularly a human IgG1 Fc domain. In some embodiments, the IgG1 Fc domain is a variant IgG1 containing D265A, N297A mutations (EU numbering) to reduce effector function.

[0331] In another embodiment, the Fc domain is an IgG4 Fc domain with reduced binding to Fc receptors. Exemplary IgG4 Fc domains with reduced binding to Fc receptors may comprise an amino acid sequence selected from Table I below. In some embodiments, the Fc domain comprises only the bolded portion of the sequence shown below:

[0332] [Table 17-1]

[0333] [Table 17-2]

[0334] [Table 17-3]

[0335] [Table 17-4]

[0336] [Table 17-5]

[0337] In certain embodiments, the IgG4 with reduced effector function comprises the bolded portion of the amino acid sequence of SEQ ID NO: 31 of WO2014 / 121087, and is sometimes referred to herein as IgG4s or hIgG4s.

[0338] For heterodimeric Fc regions, it is possible to incorporate combinations of the above-mentioned variant IgG4 Fc sequences, for example an Fc region comprising an Fc domain comprising the amino acid sequence of SEQ ID NO: 30 of WO2014 / 121087 (or a bolded portion thereof) and an Fc domain comprising the amino acid sequence of SEQ ID NO: 37 of WO2014 / 121087 (or a bolded portion thereof), or an Fc region comprising an Fc domain comprising the amino acid sequence of SEQ ID NO: 31 of WO2014 / 121087 (or a bolded portion thereof) and an Fc domain comprising the amino acid sequence of SEQ ID NO: 38 of WO2014 / 121087 (or a bolded portion thereof).

[0339] Fc Heterodimerization Variants Certain multispecific molecules, unlike native immunoglobulins, involve dimerization between two Fc domains operably linked at non-identical N-terminal regions (e.g., one Fc domain is connected to a targeting moiety (e.g., Fab) and the other Fc domain is connected to a pMHC complex). Inefficient heterodimerization of the two Fc domains to form an Fc region can be an obstacle to increasing the yield of the desired heterodimeric molecule and presents a purification challenge. Various approaches available in the art can be used to enhance dimerization of Fc domains that may be present in the multispecific molecules of the present disclosure, as disclosed, for example, in EP 1 870 459 A1, U.S. Pat. No. 5,582,996, U.S. Pat. No. 5,731,168, U.S. Pat. No. 5,910,573, U.S. Pat. No. 5,932,448, U.S. Pat. No. 6,833,441, U.S. Pat. No. 7,183,076, U.S. Pat. Appl. Publ. No. 2006 / 204493 A1, and WO 2009 / 089004 A1.

[0340] In some embodiments, the present disclosure provides Fc heterodimers, i.e., multispecific molecules comprising Fc regions comprising heterologous, non-identical Fc domains. Typically, each Fc domain in an Fc heterodimer comprises an antibody CH3 domain. The CH3 domain is derived from the constant region of an antibody of any isotype, class, or subclass, preferably the IgG (lgG1, lgG2, lgG3, and lgG4) class, as described in the preceding section.

[0341] In certain embodiments, the modification that promotes Fc heterodimer formation is a so-called "knob-into-hole" or "knob-in-hole" modification, which comprises a "knob" modification in one of the Fc domains and a "hole" modification in the other Fc domain. Knob-into-hole technology is described, for example, in U.S. Pat. Nos. 5,731,168, 7,695,936, Ridgway et al., 1996, Prot Eng 9:617-621, and Carter, 2001, Immunol Meth 248:7-15. Generally, this method involves introducing a protrusion ("knob") at the interface of a first polypeptide and a corresponding cavity ("hole") at the interface of a second polypeptide, allowing the protrusion to be positioned within the cavity, to promote heterodimer formation and impede homodimer formation. The protrusions are constructed by replacing small amino acid side chains from the interface of the first polypeptide with larger side chains (e.g., tyrosine or tryptophan). Compensatory cavities of identical or similar size to the protrusions are created in the interface of the second polypeptide by replacing the large amino acid side chains with smaller amino acid side chains (e.g., alanine or threonine).

[0342] Thus, in some embodiments, amino acid residues in the CH3 domain of a first subunit of an Fc domain are replaced with amino acid residues having a larger side chain volume, thereby creating a protrusion in the CH3 domain of the first subunit that can be positioned within a cavity in the CH3 domain of a second subunit, and amino acid residues in the CH3 domain of a second subunit of an Fc domain are replaced with amino acid residues having a smaller side chain volume, thereby creating a cavity in the CH3 domain of the second subunit into which the protrusion in the CH3 domain of the first subunit can be positioned. Preferably, the amino acid residues having a larger side chain volume are selected from the group consisting of arginine (R), phenylalanine (F), tyrosine (Y), and tryptophan (W). Preferably, the amino acid residues having a smaller side chain volume are selected from the group consisting of alanine (A), serine (S), threonine (T), and valine (V). The protrusions and cavities can be created by altering the nucleic acid encoding the polypeptide, for example, by site-directed mutagenesis or by peptide synthesis. An exemplary substitution is Y470T.

[0343] In particular such embodiments, in the first Fc domain, the threonine residue at position 366 is replaced with a tryptophan residue (T366W), and in the Fc domain, the tyrosine residue at position 407 is replaced with a valine residue (Y407V), and optionally, the threonine residue at position 366 is replaced with a serine residue (T366S), and the leucine residue at position 368 is replaced with an alanine residue (L368A) (numbering according to the Kabat EU index). In further embodiments, the first Fc domain additionally has a serine residue at position 354 replaced with a cysteine ​​residue (S354C) or a glutamic acid residue at position 356 replaced with a cysteine ​​residue (E356C) (particularly, the serine residue at position 354 is replaced with a cysteine ​​residue). The second Fc domain additionally has the tyrosine residue at position 349 replaced with a cysteine ​​residue (Y349C) (Kabat EU index numbering). In a specific embodiment, the first Fc domain comprises amino acid substitutions S354C and T366W, and the second Fc domain comprises amino acid substitutions Y349C, T366S, L368A, and Y407V (Kabat EU index numbering).

[0344] In some embodiments, electrostatic steering (e.g., as described in Gunasekaran et al., 2010, J Biol Chem 285(25):19637-46) can be used to promote association of a first Fc domain and a second Fc domain of an Fc region.

[0345] As an alternative to, or in addition to, the use of an Fc domain modified to promote heterodimerization, the Fc domain can be modified to enable a purification strategy that allows for the selection of Fc heterodimers. In one such embodiment, one polypeptide comprises a modified Fc domain that abrogates its binding to Protein A, thus enabling a purification method that results in a heterodimeric protein. See, e.g., U.S. Pat. No. 8,586,713. Thus, a multispecific molecule comprises a first CH3 domain and a second Ig CH3 domain, wherein the first Ig CH3 domain and the second Ig CH3 domain differ from each other by at least one amino acid, and the at least one amino acid difference reduces binding of the multispecific molecule to Protein A compared to a corresponding multispecific molecule lacking the amino acid difference. In one embodiment, the first CH3 domain binds to Protein A and the second CH3 domain comprises a mutation / modification, e.g., an H95R modification (according to IMGT exon numbering, H435R according to EU numbering), that reduces or eliminates Protein A binding. The second CH3 may further comprise a Y96F modification (by IMGT, Y436F by EU). This class of modifications is referred to herein as "star" mutations.

[0346] In some embodiments, the Fc may comprise one or more mutations to promote heterodimerization (e.g., knob and hole mutations) and a star mutation to facilitate purification.

[0347] Hinge Domain The multispecific molecules of the present disclosure can comprise an Fc domain comprising a hinge domain at its N-terminus. The hinge region can be a natural hinge region or a modified hinge region. The hinge region is typically found at the N-terminus of the Fc region. Unless the context dictates otherwise, the term "hinge domain" refers to a naturally occurring or non-natural hinge sequence that is a monomeric hinge domain in the context of a single or monomeric polypeptide chain, and can comprise two associated hinge sequences on separate polypeptide chains (e.g., a homodimeric or heterodimeric multispecific molecule formed by the association of two Fc domains). Sometimes, the two associated hinge sequences are referred to as a "hinge region."

[0348] A native hinge region is typically the hinge region found between the Fab and Fc domains of naturally occurring antibodies. A modified hinge region is any hinge that differs in length and / or composition from the native hinge region. Such hinges can include hinge regions from other species, such as human, mouse, rat, rabbit, shark, pig, hamster, camel, llama, or goat. Other modified hinge regions can include a complete hinge region derived from an antibody of a different class or subclass than that of the heavy chain Fc domain or Fc region. Alternatively, the modified hinge region can include a portion or repeat units of a native hinge, with each repeat unit derived from a native hinge region. In a further alternative, the native hinge region can be altered by converting one or more cysteine ​​or other residues to neutral residues such as serine or alanine, or by converting appropriately positioned residues to cysteine ​​residues. By such means, the number of cysteine ​​residues in the hinge region can be increased or decreased. Other modified hinge regions may be entirely synthetic and may be designed to have desired properties such as length, cysteine ​​composition, and flexibility.

[0349] Several modified hinge regions have been previously described, for example, in U.S. Pat. No. 5,677,425, WO99 / 15549, WO2005 / 003170, WO2005 / 003169, WO2005 / 003170, WO98 / 25971, and WO2005 / 003171, which are incorporated herein by reference.

[0350] In one embodiment, a multispecific molecule of the present disclosure comprises an Fc region in which one or both Fc domains have an intact hinge domain at their N-terminus. In various embodiments, positions 233-236 in the hinge region can be G, G, G, and empty; G, G, empty, and empty; G, empty, empty, and empty; or all empty, with positions numbered according to EU numbering.

[0351] In some embodiments, the multispecific molecules of the disclosure comprise a modified hinge region that reduces binding affinity to an Fcγ receptor compared to a wild-type hinge region of the same isotype (e.g., human IgG1 or human IgG4).

[0352] In one embodiment, a multispecific molecule of the present disclosure comprises an Fc region, wherein each Fc domain has an intact hinge domain at its N-terminus, and the Fc domain and hinge domain are each derived from IgG4, and each hinge domain comprises the modified sequence CPPC (SEQ ID NO: 133). The core hinge region of human IgG4 comprises the sequence CPSC (SEQ ID NO: 134), compared to IgG1, which comprises the sequence CPPC (SEQ ID NO: 133). The serine residues present in the IgG4 sequence provide increased flexibility in this region, and therefore a proportion of the molecules form disulfide bonds within the same protein chain (intrachain disulfides) rather than cross-linking to other heavy chains within an IgG molecule to form interchain disulfides. (Angel et al., 1993, Mol Immunol 30(1):105-108) Changing the serine residues to prolines to obtain the same core sequence as IgG1 allows for complete formation of interchain disulfides within the IgG4 hinge region, thus reducing heterogeneity in the purified product. This altered isotype is called IgG4P.

[0353] 6.7.1.3.1. Chimeric Hinge Sequences The hinge domain can be a chimeric hinge domain. For example, a chimeric hinge may comprise an "upper hinge" sequence derived from a human IgG1, human IgG2, or human IgG4 hinge region combined with a "lower hinge" sequence derived from a human IgG1, human IgG2, or human IgG4 hinge region.

[0354] In certain embodiments, the chimeric hinge region comprises the amino acid sequence EPKSCDKTHTCPPCPAPPVA (SEQ ID NO: 135, previously disclosed as SEQ ID NO: 8 in WO2014 / 121087 and incorporated herein by reference in its entirety) or ESKYGPPCPPCPAPPVA (SEQ ID NO: 136, previously disclosed as SEQ ID NO: 9 in WO2014 / 121087). Such chimeric hinge sequences may be suitably linked to an IgG4 CH2 region (e.g., by incorporation into an IgG4 Fc domain, e.g., a human Fc domain or a murine Fc domain, which may be further modified in the CH2 and / or CH3 domains to reduce effector function, e.g., as described in Section 6.7.1.1).

[0355] 6.7.1.3.2. Hinge sequences with reduced effector function In further embodiments, the hinge region can be modified to reduce effector function, for example, as described in WO2016 / 161010A2, which is incorporated herein by reference in its entirety. In various embodiments, positions 233-236 of the modified hinge region can be G, G, G, and empty; G, G, empty, and empty; G, empty, empty, and empty; or all empty, with positions numbered according to EU numbering (as shown in Figure 1 of WO2016 / 161010A2). These segments can be represented as GGG-, GG--, G---, or ----, where "-" represents an empty position.

[0356] Position 236 is vacant in canonical human IgG2 but occupied in other canonical human IgG isotypes. Positions 233-235 are occupied by residues other than G in all four human isotypes (as shown in Figure 1 of WO2016161010A2).

[0357] Hinge modifications within positions 233-236 can be combined with position 228 being occupied by P. Position 228 is naturally occupied by P in human IgG1 and IgG2, but is occupied by S in human IgG4 and by R in human IgG3. The S228P mutation in IgG4 antibodies is advantageous for stabilizing IgG4 antibodies and reducing heavy-light chain pair exchange between exogenous and endogenous antibodies. Preferably, positions 226-229 are occupied by C, P, P, and C, respectively.

[0358] Exemplary hinge regions have residues 226-236, sometimes referred to as the middle (or core) and lower hinge, occupied by modified hinge sequences designated GGG-(233-236), GG--(233-236), G---(233-236), and no G(233-236). Optionally, the hinge domain amino acid sequence comprises CPPCPAPGGG-GPSVF (SEQ ID NO: 137, previously disclosed as SEQ ID NO: 1 in WO2016 / 161010A2), CPPCPAPGG--GPSVF (SEQ ID NO: 138, previously disclosed as SEQ ID NO: 2 in WO2016 / 161010A2), CPPCPAPG---GPSVF (SEQ ID NO: 139, previously disclosed as SEQ ID NO: 3 in WO2016 / 161010A2), or CPPCPAP----GPSVF (SEQ ID NO: 140, previously disclosed as SEQ ID NO: 4 in WO2016 / 161010A2).

[0359] The modified hinge regions described above can be incorporated into heavy chain constant regions, which typically include a CH2 domain and a CH3 domain and may have additional hinge segments (e.g., upper hinges) adjacent to the designated regions. The additional constant region segments typically have the same isotype, preferably a human isotype, but may also be hybrids of different isotypes. The isotype of such additional human constant region segments is preferably human IgG4, but may also be human IgG1, IgG2, or IgG3, or hybrids thereof, in which the domains are of different isotypes. Exemplary sequences of human IgG1, IgG2, and IgG4 are shown in Figures 2-4 of WO2016 / 161010A2.

[0360] In certain embodiments, a modified hinge sequence can be linked to an IgG4 CH2 region (which can be further modified in the CH2 and / or CH3 domains to reduce effector function, e.g., by incorporation into an IgG4 Fc domain, e.g., a human Fc domain or a mouse Fc domain, e.g., as described in Section 6.7.1.1).

[0361] Linker In certain aspects, the present disclosure provides multispecific molecules (e.g., T cell activators) in which two or more components of the multispecific molecule are connected to each other by a peptide linker. By way of example and not limitation, a linker can be used to connect (a) a pMHC complex and a multimerization moiety, (b) a pMHC complex and an immune cell antigen targeting moiety, (c) an immune cell antigen targeting moiety and a multimerization moiety (e.g., an Fab domain and an Fc domain), (d) a tumor antigen targeting moiety and a multimerization moiety (e.g., an Fab domain and an Fc domain), (e) different components of a pMHC complex (e.g., an antigenic peptide and an MHC molecule), or (f) different domains within an immune cell antigen targeting moiety (e.g., the VH and VL domains within an scFv).

[0362] The peptide linker can range from 2 to 60 or more amino acids, and in certain embodiments, the peptide linker can range from 3 to 50 amino acids, 4 to 30 amino acids, 5 to 25 amino acids, 10 to 25 amino acids, 10 to 60 amino acids, 12 to 20 amino acids, 20 to 50 amino acids, or 25 to 35 amino acids in length.

[0363] In certain embodiments, the peptide linker is at least 5 amino acids, at least 6 amino acids, or at least 7 amino acids in length, and optionally up to 30 amino acids, up to 40 amino acids, up to 50 amino acids, or up to 60 amino acids in length.

[0364] In some of the foregoing embodiments, the linker is between 5 and 50 amino acids in length, e.g., between 5 and 50, 5 and 45, 5 and 40, 5 and 35, 5 and 30, 5 and 25, or 5 and 20 amino acids in length. In other of the foregoing embodiments, the linker is between 6 and 50 amino acids in length, e.g., between 6 and 50, 6 and 45, 6 and 40, 6 and 35, 6 and 30, 6 and 25, or 6 and 20 amino acids in length. In still other of the foregoing embodiments, the linker is between 7 and 50 amino acids in length, e.g., between 7 and 50, 7 and 45, 7 and 40, 7 and 35, 7 and 30, 7 and 25, or 7 and 20 amino acids in length.

[0365] Charged (eg, charged hydrophilic linkers) and / or flexible linkers are particularly preferred. Examples of flexible linkers that can be used in the multispecific molecules of the present disclosure include those disclosed in Chen et al., 2013, Adv Drug Deliv Rev. 65(10):1357-1369, and Klein et al., 2014, Protein Engineering, Design & Selection 27(10):325-330. Particularly useful flexible linkers include repeats of glycine and serine, e.g., G n S (SEQ ID NO: 141) or SG n(SEQ ID NO: 142), where n is an integer from 1 to 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In one embodiment, the linker is a G4S (SEQ ID NO: 143), e.g., (GGGGS) n (SEQ ID NO: 144), or includes a repeating monomer or polymer thereof, where n is an integer of 1 to 10.

[0366] Polyglycine linkers are suitable for use in the T cell activators of the present disclosure. In some embodiments, the peptide linker comprises two consecutive glycines (2 Gly), three consecutive glycines (3 Gly), four consecutive glycines (4 Gly) (SEQ ID NO: 145), five consecutive glycines (5 Gly) (SEQ ID NO: 146), six consecutive glycines (6 Gly) (SEQ ID NO: 147), seven consecutive glycines (7 Gly) (SEQ ID NO: 148), eight consecutive glycines (8 Gly) (SEQ ID NO: 149), or nine consecutive glycines (9 Gly) (SEQ ID NO: 150).

[0367] Linkers of the present disclosure (e.g., linkers between a pMHC complex and an Fc domain, linkers between an immune cell antigen targeting moiety and an Fc domain, etc.) may, in some embodiments, be "short" linkers (i.e., linkers seven amino acids or less in length). A short linker may be at most or exactly 7, 6, 5, 4, 3, or 2 amino acids in length. In some embodiments, a T cell activator of the present disclosure comprises a pMHC complex and an Fc domain operably linked by a short linker. Alternatively, linkers of the present disclosure may, in some embodiments, be "long" linkers (i.e., linkers greater than seven amino acids in length). A long linker may be at least or exactly 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length, or more. In some embodiments, a T cell activator of the present disclosure comprises a pMHC complex and an Fc domain operably linked by a long linker.

[0368] Exemplary linker sequences are set forth in Table L below. A T cell activator of the present disclosure may comprise one or more linkers of Table L.

[0369] [Table 18-1]

[0370] [Table 18-2]

[0371] [Table 18-3]

[0372] In some embodiments, the T cell activator comprises linker L1. In some embodiments, the T cell activator comprises linker L2. In some embodiments, the T cell activator comprises linker L3. In some embodiments, the T cell activator comprises linker L4. In some embodiments, the T cell activator comprises linker L5. In some embodiments, the T cell activator comprises linker L6. In some embodiments, the T cell activator comprises linker L7. In some embodiments, the T cell activator comprises linker L8. In some embodiments, the T cell activator comprises linker L9. In some embodiments, the T cell activator comprises linker L10. In some embodiments, the T cell activator comprises linker L11. In some embodiments, the T cell activator comprises linker L12. In some embodiments, the T cell activator comprises linker L13. In some embodiments, the T cell activator comprises linker L14. In some embodiments, the T cell activator comprises linker L15. In some embodiments, the T cell activator comprises linker L16. In some embodiments, the T cell activator comprises linker L17. In some embodiments, the T cell activator comprises linker L18. In some embodiments, the T cell activator comprises linker L19. In some embodiments, the T cell activator comprises linker L20. In some embodiments, the T cell activator comprises linker L21. In some embodiments, the T cell activator comprises linker L22. In some embodiments, the T cell activator comprises linker L23. In some embodiments, the T cell activator comprises linker L24. In some embodiments, the T cell activator comprises linker L25. In some embodiments, the T cell activator comprises linker L26. In some embodiments, the T cell activator comprises linker L27. In some embodiments, the T cell activator comprises linker L28. In some embodiments, the T cell activator comprises linker L29. In some embodiments, the T cell activator comprises linker L30. In some embodiments, the T cell activator comprises linker L31.In some embodiments, the T cell activator comprises linker L32. In some embodiments, the T cell activator comprises linker L33. In some embodiments, the T cell activator comprises linker L34. In some embodiments, the T cell activator comprises linker L35. In some embodiments, the T cell activator comprises linker L36. In some embodiments, the T cell activator comprises linker L37. In some embodiments, the T cell activator comprises linker L38. In some embodiments, the T cell activator comprises linker L39. In some embodiments, the T cell activator comprises linker L40. In some embodiments, the T cell activator comprises linker L41. In some embodiments, the T cell activator comprises linker L42. In some embodiments, the T cell activator comprises linker L43. In some embodiments, the T cell activator comprises linker L44. In some embodiments, the T cell activator comprises linker L45. In some embodiments, the T cell activator comprises linker L46. In some embodiments, the T cell activator comprises linker L47. In some embodiments, the T cell activator comprises linker L48. In some embodiments, the T cell activator comprises linker L49. In some embodiments, the T cell activator comprises linker L50. In some embodiments, the T cell activator comprises linker L51. In some embodiments, the T cell activator comprises linker L52. In some embodiments, the T cell activator comprises linker L53. In some embodiments, the T cell activator comprises linker L54. In some embodiments, the T cell activator comprises linker L55. In some embodiments, the T cell activator comprises linker L56. In some embodiments, the T cell activator comprises linker L57. In some embodiments, the T cell activator comprises linker L58. In some embodiments, the T cell activator comprises linker L59. In some embodiments, the T cell activator comprises linker L60. In some embodiments, the T cell activator comprises linker L61. In some embodiments, the T cell activator comprises linker L62.In some embodiments, the T cell activator comprises linker L63. In some embodiments, the T cell activator comprises linker L64. In some embodiments, the T cell activator comprises linker L65. In some embodiments, the T cell activator comprises linker L66. In some embodiments, the T cell activator comprises linker L67. In some embodiments, the T cell activator comprises linker L68. In some embodiments, the T cell activator comprises linker L69. In some embodiments, the T cell activator comprises linker L70. In some embodiments, the T cell activator comprises linker L71. In some embodiments, the T cell activator comprises linker L72. In some embodiments, the T cell activator comprises linker L73. In some embodiments, the T cell activator comprises linker L74. In some embodiments, the T cell activator comprises linker L75. In some embodiments, the T cell activator comprises linker L76. In some embodiments, the T cell activator comprises linker L77. In some embodiments, the T cell activator comprises linker L78. In some embodiments, the T cell activator comprises the linker L79.

[0373] 6.8.1. pMHC Linker In pMHC complexes, suitable linkers can range from 1 amino acid (e.g., Gly) to 20 amino acids, 2 to 15 amino acids, 3 to 12 amino acids, including 4 to 10 amino acids, 5 to 9 amino acids, 6 to 8 amino acids, or 7 to 8 amino acids, and can be 1, 2, 3, 4, 5, 6, or 7 amino acids. In addition to the linkers described above, pMHC linkers include glycine polymers (G)n, glycine-serine polymers (e.g., (GS)n, (GSGGS)n (SEQ ID NO: 158), and (GGGS)n (SEQ ID NO: 152), where n is at least one integer), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers can be used. Both Gly and Ser are relatively unstructured and can function as neutral tethers between components. Glycine polymers can be used. Glycine has access to much more phi-psi space than alanine and is much less restricted than residues with longer side chains (see Scheraga, 1992, Rev. Computational Chem. 1 1173-142, the entire contents of which are incorporated herein by reference). Exemplary linkers can include amino acid sequences including, but not limited to, GGSG (SEQ ID NO: 184), GGSGG (SEQ ID NO: 185), GSGSG (SEQ ID NO: 197), GSGGG (SEQ ID NO: 196), GGGSG (SEQ ID NO: 182), GSSSG (SEQ ID NO: 206), GCGASGGGGSGGGGS (SEQ ID NO: 227), GGGGSGGGGS (SEQ ID NO: 151), GGGASGGGGSGGGGS (SEQ ID NO: 228), GGGGSGGGGSGGGGS (SEQ ID NO: 127), GGGASGGGGS (SEQ ID NO: 229), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 179), GCGGS (SEQ ID NO: 230), etc. In some embodiments, the linker polypeptide comprises a cysteine ​​residue that can form a disulfide bond with a cysteine ​​residue present in another portion of the pMHC complex. In certain embodiments, the linker comprises the amino acid sequence GCGGS (SEQ ID NO: 230).Substitution of glycine with cysteine ​​in the G4S (SEQ ID NO: 143) linker can result in the formation of a disulfide bond, e.g., an MHC targeting moiety with the corresponding cysteine ​​substitution in HLA.A2, which stabilizes the MHC peptide in the MHC complex.

[0374] 6.9. Nucleic Acids and Host Cells In another aspect, the present disclosure provides nucleic acids encoding the multispecific molecules (e.g., T cell activators) of the present disclosure. In some embodiments, the multispecific molecules are encoded by a single nucleic acid. In other embodiments, for example, in the case of heterodimeric molecules or molecules that include components composed of more than one polypeptide chain (e.g., pMHC complexes, immune cell antigen targeting moieties), the multispecific molecules can be encoded by multiple (e.g., two, three, four, or more) nucleic acids.

[0375] A single nucleic acid can encode a multispecific molecule comprising a single polypeptide chain, a multispecific molecule comprising two or more polypeptide chains, or a portion of a multispecific molecule comprising three or more polypeptide chains (e.g., a single nucleic acid can encode two polypeptide chains of a multispecific molecule comprising three, four, or more polypeptide chains, or three polypeptide chains of a multispecific molecule comprising four or more polypeptide chains). To separately control expression, open reading frames encoding two or more polypeptide chains can be under the control of separate transcriptional regulatory elements (e.g., promoters and / or enhancers). Open reading frames encoding two or more polypeptides can also be controlled by the same transcriptional regulatory element and separated by an internal ribosome entry site (IRES) sequence, allowing translation into separate polypeptides.

[0376] In some embodiments, multispecific molecules comprising two or more polypeptide chains are encoded by two or more nucleic acids. The number of nucleic acids encoding the multispecific molecule can be equal to or less than the number of polypeptide chains in the multispecific molecule (e.g., when two or more polypeptide chains are encoded by a single nucleic acid).

[0377] The nucleic acids of the present disclosure can be DNA (e.g., a plasmid) or RNA (e.g., mRNA). In some embodiments, the present disclosure provides methods for delivering a T cell activator to an individual in need thereof by administering a nucleic acid of the present disclosure. The nucleic acid can be delivered to an individual via a variety of pharmaceutical compositions, including prophylactic compositions, examples of which are described in Section 6.10.

[0378] In another aspect, the disclosure provides host cells and vectors comprising the nucleic acids of the disclosure. The nucleic acids may be present in a single vector or may be present in separate vectors present in the same host cell or in separate host cells, as described in more detail herein below.

[0379] Vectors The present disclosure provides vectors comprising nucleotide sequences encoding one or two of the polypeptide chains of the multispecific molecules or multispecific molecule components described herein, e.g., T cell activators. Vectors include, but are not limited to, viruses, plasmids, cosmids, lambda phage, or yeast artificial chromosomes (YACs). Vectors encoding the T cell activators (or components thereof) of the present disclosure can be useful in expressing and / or delivering the T cell activators.

[0380] Numerous vector systems can be used. For example, one class of vectors utilizes DNA elements derived from animal viruses such as bovine papillomavirus, polyomavirus, adenovirus, vaccinia virus, baculovirus, retrovirus (Rous sarcoma virus, MMTV, or MOMLV), or SV40 virus. Another class of vectors utilizes RNA elements derived from RNA viruses such as Semliki Forest virus, eastern equine encephalitis virus, and flaviviruses.

[0381] Additionally, cells that have stably integrated the DNA into their chromosomes can be selected by introducing one or more markers that allow for the selection of transfected host cells. Markers can provide, for example, prototropy to auxotrophic hosts, biocide resistance (e.g., antibiotics), or resistance to heavy metals such as copper. The selectable marker gene can be either directly linked to the DNA sequence to be expressed or introduced into the same cell by cotransformation. Additional elements may also be required for optimal mRNA synthesis. These elements may include splice signals, as well as transcription promoters, enhancers, and termination signals.

[0382] When the DNA sequence comprising expression vector or construct is prepared for expression, expression vector can be transfected or introduced into suitable host cell.To achieve this, various techniques can be used, such as protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene gun, lipid-based transfection or other conventional techniques.The method and conditions for culturing the resulting transfected cells and recovering expressed polypeptide are known to those skilled in the art, and can be changed or optimized according to the specific expression vector and mammalian host cell used based on this specification.

[0383] 6.9.2.Host cells The present disclosure also provides a host cell comprising a nucleic acid of the present disclosure. In one embodiment, the host cell is genetically engineered to contain one or more nucleic acids described herein.

[0384] In one embodiment, the host cell is genetically engineered using an expression cassette. The term "expression cassette" refers to a nucleotide sequence that can affect the expression of a gene in a host compatible with such a sequence. Such a cassette can include a promoter, an open reading frame with or without introns, and a termination signal. Additional factors necessary or useful for effecting expression, such as an inducible promoter, can also be used.

[0385] The present disclosure also provides host cells comprising the vectors described herein. Host cells can be, but are not limited to, eukaryotic cells, bacterial cells, insect cells, or human cells. Suitable eukaryotic cells include, but are not limited to, Vero cells, HeLa cells, COS cells, CHO cells, HEK293 cells, BHK cells, and MDCKII cells. Suitable insect cells include, but are not limited to, Sf9 cells.

[0386] Pharmaceutical Compositions 6.10.1. Pharmaceutical Compositions Comprising T Cell Activator Polypeptides The T cell activator of the present disclosure may be in the form of a composition comprising the T cell activator and one or more carriers, excipients, and / or diluents. The composition may be formulated for a particular use, such as veterinary use or pharmaceutical use in humans. The form of the composition (e.g., dry powder, liquid formulation, etc.) and the excipients, diluents, and / or carriers used will depend on the intended use of the T cell activator and, in the case of therapeutic applications, the mode of administration.

[0387] For therapeutic use, the composition may be supplied as part of a sterile pharmaceutical composition containing a pharmaceutically acceptable carrier. This composition may be in any suitable form (depending on the desired method of administration to a patient). Pharmaceutical compositions may be administered to a patient by a variety of routes, including oral, transdermal, subcutaneous, intranasal, intravenous, intramuscular, intratumoral, intrathecal, local, or topical. The most suitable route for administration in any given case will depend on the particular antibody, the subject, and the nature and severity of the disease, as well as the physical condition of the subject. Typically, pharmaceutical compositions will be administered intravenously or subcutaneously.

[0388] The pharmaceutical composition can be conveniently presented in a unit dosage form containing a predetermined amount of the T cell activator of the present disclosure per dose. The amount of T cell activator contained in the unit dose will depend on the disease being treated and other factors well known in the art. Such a unit dosage can be in the form of a lyophilized dry powder containing an amount of T cell activator suitable for a single administration, or in liquid form. The dry powder unit dosage form can be packaged in a kit together with a syringe, a suitable amount of diluent, and / or other components useful for administration. The liquid unit dosage can be conveniently provided in the form of a syringe pre-filled with an amount of T cell activator suitable for a single administration.

[0389] The pharmaceutical compositions may also be supplied in bulk, as they contain amounts of T cell activators suitable for multiple administration. Pharmaceutical compositions can be prepared for storage as lyophilized formulations or aqueous solutions by mixing a T cell activator having the desired purity with any pharmaceutically acceptable carrier, excipient, or stabilizer (all of which are referred to herein as "carriers") typically used in the art, i.e., buffers, stabilizers, preservatives, tonicity agents, non-ionic detergents, antioxidants, and various other additives. See Remington's Pharmaceutical Sciences, 16th edition (Osol, ed. 1980). Such additives should be nontoxic to the recipient at the dosages and concentrations employed.

[0390] Buffering agents help maintain pH in a range close to physiological conditions. They can be present in a wide variety of concentrations, but will typically be present at concentrations ranging from about 2 mM to about 50 mM. Suitable buffering agents for use in the present disclosure include both organic and inorganic acids and their salts, such as citrate buffers (e.g., monosodium citrate-disodium citrate mixtures, citric acid-trisodium citrate mixtures, citric acid-monosodium citrate mixtures, etc.), succinate buffers (e.g., succinic acid-monosodium succinate mixtures, succinic acid-sodium hydroxide mixtures, succinic acid-disodium succinate mixtures, etc.), tartrate buffers (e.g., tartaric acid-sodium tartrate mixtures, tartaric acid-potassium tartrate mixtures, tartaric acid-sodium hydroxide mixtures, etc.), fumarate buffers (e.g., fumaric acid-monosodium fumarate mixtures, disodium fumarate mixtures, monosodium fumarate-disodium fumarate mixtures, etc.), gluconate buffers (e.g., gluconate-sodium glyconate mixtures, gluconate-sodium hydroxide mixtures, gluconate-potassium gluconate mixtures, etc.), glyuconate mixtures, etc.), oxalate buffers (e.g., oxalic acid-sodium oxalate mixtures, oxalic acid-sodium hydroxide mixtures, oxalic acid-potassium oxalate mixtures, etc.), lactate buffers (e.g., lactic acid-sodium lactate mixtures, lactic acid-sodium hydroxide mixtures, lactic acid-potassium lactate mixtures, etc.), and acetate buffers (e.g., acetic acid-sodium acetate mixtures, acetic acid-sodium hydroxide mixtures, etc.). Additionally, phosphate buffers, histidine buffers, and trimethylamine salts (e.g., Tris) can be used.

[0391] Preservatives may be added to retard microbial growth and can be added in amounts ranging from about 0.2% to 1% (w / v). Suitable preservatives for use in the present disclosure include phenol, benzyl alcohol, meta-cresol, methylparaben, propylparaben, octadecyldimethylbenzylammonium chloride, benzalconium halides (e.g., chloride, bromide, and iodide), hexamethonium chloride, alkylparabens (e.g., methyl or propylparaben), catechol, resorcinol, cyclohexanol, and 3-pentanol. Tonicity adjusting agents, sometimes known as "stabilizers," may be added to ensure the isotonicity of the liquid compositions of the present disclosure and include polyhydric sugar alcohols, such as trihydric or higher sugar alcohols (e.g., glycerin, erythritol, arabitol, xylitol, sorbitol, and mannitol). Stabilizers refer to a broad category of excipients that can range in function from bulking agents to additives, and serve to solubilize the therapeutic agent or prevent it from denaturing or adhering to the container wall.Typical stabilizers include polyhydric sugar alcohols (as listed above), amino acids (e.g., arginine, lysine, glycine, glutamine, asparagine, histidine, alanine, ornithine, L-leucine, 2-phenylalanine, glutamic acid, threonine, etc.), organic sugars or sugar alcohols (e.g., lactose, trehalose, stachyose, mannitol, sorbitol, xylitol, ribitol, myoinisitol, galactitol, glycerol, etc., including cyclitols such as inositol), polyethylene glycol, amino acid polymers, sulfur-containing reducing agents (e.g., urea, glutathione, Stabilizers can be selected from the group consisting of thioctic acid, sodium thioglycolate, thioglycerol, α-monothioglycerol, and sodium thiosulfate, low molecular weight polypeptides (e.g., peptides of 10 residues or less), proteins (e.g., human serum albumin, bovine serum albumin, gelatin, or immunoglobulins), hydrophilic polymers (e.g., polyvinylpyrrolidone), monosaccharides (e.g., xylose, mannose, fructose, glucose), disaccharides (e.g., lactose, maltose, sucrose, and trehalose), trisaccharides (e.g., raffinose), and polysaccharides (e.g., dextran). The stabilizer can be present in an amount ranging from 0.5 to 10% by weight of the T cell activator.

[0392] Nonionic surfactants or detergents (also known as "wetting agents") can be added to aid in solubilizing the glycoprotein and to protect it from agitation-induced aggregation, allowing the formulation to be exposed to stressful shear surfaces without denaturing the protein. Suitable nonionic surfactants include polysorbates (e.g., 20, 80), polyoxamers (e.g., 184, 188), and Pluronic® polyols. The nonionic surfactant may be present in a range of about 0.05 mg / mL to about 1.0 mg / mL (e.g., about 0.07 mg / mL to about 0.2 mg / mL).

[0393] Additional miscellaneous excipients include bulking agents (eg, starch), chelating agents (eg, EDTA), antioxidants (eg, ascorbic acid, methionine, vitamin E), and cosolvents.

[0394] 6.10.2. Pharmaceutical Compositions for Delivering Nucleic Acid-Encoding T Cell Activators The T cell activators of the present disclosure can be delivered by nucleic acid encoding the T cell activator, for example, as a plasmid, DNA, mRNA, or via a viral vector encoding the T cell activator under the control of a suitable promoter.

[0395] Exemplary vectors include adenovirus or AAV-based therapeutics. Non-limiting examples of adenovirus- or AAV-based therapeutics for use in the methods, uses, or compositions herein include, for example, rAd-p53 (also known as Gendicine®, Genkaxin®), a recombinant adenovirus vector encoding wild-type human tumor suppressor protein p53, for use in the treatment of cancer; Ad5_d11520 (also known as H101 or ONYX-015), an adenovirus lacking the E1B gene to inactivate host p53; see, e.g., Russell et al., 2012, Nature Biotechnology 30:658-670); AD5-D24-GM-CSF (Cerullo et al., 2010, Cancer Res. 70:4297; rAd-HSVtk, a replication-deficient adenovirus carrying the HSV thymidine kinase gene, for example, for the treatment of cancer (Cerepro®, developed by Ark Therapeutics, see, e.g., U.S. Pat. No. 6,579,855; developed by Advantagene as ProstAtak™; see International Publication WO 2005 / 049094); rAd-TNFα, a replication-deficient adenovirus vector expressing human tumor necrosis factor alpha (TNFα) under the control of the chemoradiation-inducible EGR-1 promoter, for example, for the treatment of cancer (TNFerade™, GenVec; Rasmussen et al., 2002, Cancer Gene Ther. 9:951-7; for example, Ad-IFNβ (BG00001 and H5.110CMVhIFN-β, Biogen), an adenovirus serotype 5 vector with deleted E1 and E3 genes that expresses the human interferon-beta gene under the direction of the cytomegalovirus (CMV) immediate early promoter for the treatment of cancer.(2010, Mol. Ther. 18:852-860). Additional vectors are recognized in the art and include, for example, lentiviral vectors (e.g., VSV), retroviral vectors, and other vectors.

[0396] Any currently known or future developed delivery vector, natural or engineered, can be used to deliver the T cell activators of the present disclosure. In some embodiments, the delivery vector is a viral vector, e.g., comprising a virus, a viral capsid, a viral genome, etc. In some embodiments, the delivery vector is a naked nucleic acid, e.g., an episome. In some embodiments, the delivery vector comprises a nucleic acid complex. Exemplary non-limiting nucleic acid complexes for use as delivery vectors include lipoplexes, polymersomes, polypex, dendrimers, inorganic nanoparticles (e.g., polynucleotide-coated gold, silica, iron oxide, calcium phosphate, etc.). In some embodiments, the delivery vectors described herein comprise a combination of viral vectors, naked nucleic acids, and nucleic acid complexes.

[0397] In one embodiment, the delivery vector is a virus, including a retrovirus, adenovirus, herpes simplex virus, poxvirus, vaccinia virus, lentivirus, or adeno-associated virus. In one embodiment, the delivery vector is an adeno-associated virus (AAV), including serotypes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, and AAV11, or an engineered or naturally selected variant thereof.

[0398] In one embodiment, the nucleic acid encoding the T cell activator (or a component thereof) also contains an adeno-associated virus (AAV) nucleic acid sequence. In one embodiment, the vector is a chimeric adeno-associated virus containing genetic elements from more than one serotype. For example, an AAV vector having the rep gene from AAV1 and the cap gene from AAV2 (designated as AAV1 / 2 or AAV RC1 / 2) can be used as a delivery vector to deliver the T cell activator expressing the nucleic acid to cells in a patient or cells in need thereof.In one embodiment, the delivery vector is AAV1 / 2, AAV1 / 3, AAV1 / 4, AAV1 / 5, AAV1 / 6, AAV1 / 7, AAV1 / 8, AAV1 / 9, AAV1 / 10, AAV1 / 11, AAV2 / 1, AAV2 / 3, AAV2 / 4, AAV2 / 5, AAV2 / 6, AAV2 / 7, AAV2 / 8, AAV2 / 9, AAV2 / 10, AAV2 / 11, AAV3 / 1, AAV3 / 2, AAV3 / 4, AAV3 / 5, AAV3 / 6, AAV3 / 7, AAV3 / 8, AAV3 / 9, AAV3 / 10, AAV3 / 10, AAV4 / 1, AAV4 / 2, AAV4 / 3, AAV4 / 5, AAV4 / 6, AAV4 / 7, AAV4 / 8, AAV4 / 9, AAV4 / 10, AAV4 / 11, AAV5 / 1, AAV5 / 2, A AV5 / 3, AAV5 / 4, AAV5 / 6, AAV5 / 7, AAV5 / 8, AAV5 / 9, AAV5 / 10, AAV5 / 11, AAV6 / 1, AAV6 / 2, AAV6 / 3, AAV6 / 4, AAV6 / 5, AAV6 / 7, AAV6 / 8, A AV6 / 9, AAV6 / 10, AAV6 / 10, AAV7 / 1, AAV7 / 2, AAV7 / 3, AAV7 / 4, AAV7 / 5, AAV7 / 6, AAV7 / 8, AAV7 / 9, AAV7 / 10, AAV7 / 11, AAV8 / 1, AAV8 / 2, AAV8 / 3, AAV8 / 4, AAV8 / 5, AAV8 / 6, AAV8 / 7, AAV8 / 9, AAV8 / 10, AAV8 / 11, AAV9 / 1, AAV9 / 2, AAV9 / 3, AAV9 / 4, AAV9 / 5, AAV9 / 6, AAV9 / 7, AAV9 / 8, AAV9 / 10, AAV9 / 11, AAV10 / 1, AAV10 / 2, AAV10 / 3, AAV10 / 4, AAV10 / 5, AAV10 / 6, AAV10 / 7, AAV10 / 8, AAV10 / 9, AAV10 / 11, AAV11 / 1, AAV11 / 2, AAV11 / 3, AAV11 / 4, AAV11 / 5, AAV11 / 6, AAV11 / 7, AAV11 / 8, AAV11 / 9, AAV11 / 10, chimeric viral vectors, or derivatives thereof.Gao et al., "Novel adeno-associated viruses from rhesus monkeys as vectors for human gene therapy," PNAS 99(18):11854-11859, September 3, 2002, is incorporated herein by reference for its discussion of AAV and chimeric viral vectors useful as delivery vectors, and their construction and use.

[0399] Nucleic acid preparations Nucleic acids of the present disclosure (e.g., encoding T cell activators or components thereof) can be formulated or administered in combination with one or more pharmaceutically acceptable excipients. In addition to conventional excipients such as any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersing or suspending aids, surfactants, isotonicity agents, thickening or emulsifying agents, and preservatives, excipients can include, but are not limited to, lipidoids, liposomes, lipid nanoparticles, polymers, lipoplexes, core-shell nanoparticles, peptides, proteins, cells transfected with nucleic acids (e.g., for transplantation into a subject), hyaluronidase, nanoparticle mimics, and combinations thereof.

[0400] In some embodiments, the nucleic acids disclosed herein (i.e., nucleic acids encoding T cell activators or components thereof) can be formulated as nanoparticles. Nanoparticle compositions are typically sized on the order of micrometers or smaller and can contain a lipid bilayer. Nanoparticle compositions encompass lipid nanoparticles (LNPs), liposomes (e.g., lipid vesicles), and lipoplexes. For example, the nanoparticle composition can be a liposome having a lipid bilayer with a diameter of 500 nm or less. Accordingly, the present disclosure also provides nanoparticle compositions comprising (i) a lipid composition comprising a delivery agent and (ii) one or more nucleic acids encoding the T cell activators or components thereof disclosed herein. In such nanoparticle compositions, the lipid compositions disclosed herein can encapsulate the nucleic acid(s). In some embodiments, the nanoparticle composition is a vesicle comprising one or more lipid bilayers. In certain embodiments, the nanoparticle composition comprises two or more concentric bilayers separated by aqueous compartments. The lipid bilayers can be functionalized and / or crosslinked to each other. The lipid bilayers can comprise one or more ligands, proteins, or channels. In one embodiment, the lipid nanoparticle comprises an ionizable lipid, a structured lipid, a phospholipid, and mRNA. In some embodiments, the LNP comprises an ionizable lipid, a PEG-modified lipid, a phospholipid, and a structured lipid.

[0401] In some embodiments, the nucleic acids described herein include complexes such as, but not limited to, nanoparticles (e.g., polynucleotide self-assembled nanoparticles, polymer-based self-assembled nanoparticles, inorganic nanoparticles, lipid nanoparticles, semiconductor / metal nanoparticles), gels and hydrogels, polynucleotide complexes with cations and anions, microparticles, and any combination thereof.

[0402] In some embodiments, the nucleic acids disclosed herein can be formulated as self-assembled nanoparticles. As a non-limiting example, the nucleic acids can be used to create nanoparticles that can be used in nucleic acid delivery systems (see, for example, International Publication No. WO2012 / 125987, the entire contents of which are incorporated herein by reference). In some embodiments, the nucleic acid self-assembled nanoparticles can include a nucleic acid core disclosed herein and a polymer shell. The polymer shell can be any of the polymers described herein and known in the art. In additional embodiments, the polymer shell can be used to protect the nucleic acid in the core.

[0403] In some embodiments, these self-assembled nanoparticles may be microsponges formed from long polymers of nucleic acid hairpins that form crystalline "pleated" sheets before self-assembling into microsponges. These microsponges are densely packed, sponge-like microparticles that can function as efficient carriers and deliver cargo to cells. Microsponges can be 1 μm to 300 nm in diameter. Microsponges can be complexed with other drugs known in the art to form larger microsponges. As a non-limiting example, microsponges can be complexed with drugs to form an outer layer to promote cellular uptake, such as the polycation polyethyleneimine (PEI). This complex can form 250 nm diameter particles that can be stably maintained at high temperatures (150°C) (Grabow and Jaegar, Nature Materials 2012, 11:269-269, incorporated herein by reference in its entirety). Furthermore, these microsponges may be capable of exhibiting an exceptional degree of protection from degradation by ribonucleases. In another embodiment, polymer-based self-assembled nanoparticles, such as, but not limited to, microsponges, can be fully programmable nanoparticles: the shape, size, and stoichiometry of the nanoparticles can be precisely controlled to generate nanoparticles optimized for delivery of cargo, such as, but not limited to, nucleic acids.

[0404] In some embodiments, nucleic acids can be formulated into inorganic nanoparticles (see U.S. Patent No. 8,257,745, which is incorporated herein by reference in its entirety). Inorganic nanoparticles can include, but are not limited to, water-swellable clay materials. As a non-limiting example, inorganic nanoparticles can include synthetic smectite clays made from simple silicates (see U.S. Patent Nos. 5,585,108 and 8,257,745, each of which is incorporated herein by reference in its entirety).

[0405] In some embodiments, nucleic acids may be formulated into water-dispersible nanoparticles comprising semiconductor or metallic materials (U.S. Patent Application Publication No. 2012 / 0228565, which is incorporated herein by reference in its entirety), or formed into magnetic nanoparticles (U.S. Patent Application Publication Nos. 2012 / 0265001 and 2012 / 0283503, each of which is incorporated herein by reference in its entirety). Water-dispersible nanoparticles can be hydrophobic or hydrophilic.

[0406] In some embodiments, the nucleic acids disclosed herein can be encapsulated in any hydrogel known in the art that can form a gel when injected into a subject. Hydrogels are networks of hydrophilic polymer chains and are often found as colloidal gels in which water is the dispersion medium. Hydrogels are highly absorbent (capable of containing 99% or more water) natural or synthetic polymers. Due to their significant water content, hydrogels also have a degree of flexibility very similar to that of natural tissue. The hydrogels described herein can be used to encapsulate lipid nanoparticles that are biocompatible, biodegradable, and / or porous. As a non-limiting example, the hydrogel can be an aptamer-functionalized hydrogel. The aptamer-functionalized hydrogel can be programmed to release one or more polynucleotides using polynucleotide hybridization. (Battig et al., J. Am. Chem. Society. 2012 134:12410-12413, incorporated herein by reference in its entirety.) In some embodiments, the polynucleotide may be encapsulated in a lipid nanoparticle, which may then be encapsulated in a hydrogel.

[0407] In some embodiments, the nucleic acids disclosed herein can be encapsulated in fibrin gels, fibrin hydrogels, or fibrin glue. In another embodiment, the nucleic acids can be formulated into lipid nanoparticles or rapidly removed lipid nanoparticles before being encapsulated in fibrin gels, fibrin hydrogels, or fibrin glue. In yet another embodiment, the nucleic acids can be formulated as lipoplexes before being encapsulated in fibrin gels, hydrogels, or fibrin glue. Fibrin gels, hydrogels, and glues contain two components: a fibrinogen solution and a calcium-enriched thrombin solution (see, e.g., Spicer and Mikos, Journal of Controlled Release 2010, 148:49-55; Kidd et al., Journal of Controlled Release 2012, 157:80-85, each of which is incorporated herein by reference in its entirety). The concentrations of the components of the fibrin gel, hydrogel, and / or adhesive can be varied to alter the properties, network mesh size, and / or degradation characteristics of the gel, hydrogel, and / or adhesive, including, but not limited to, altering the release characteristics of the fibrin gel, hydrogel, and / or adhesive (see, e.g., Spicer and Mikos, Journal of Controlled Release 2010. 148:49-55; Kidd et al. Journal of Controlled Release 2012. 157:80-85; Catelas et al. Tissue Engineering 2008. 14:119-128, each of which is incorporated herein by reference in its entirety).

[0408] In some embodiments, the nucleic acid disclosed herein can contain cations or anions.In one embodiment, the formulation includes, but is not limited to, metal cations such as Zn2+, Ca2+, Cu2+, Mg+, and combinations thereof.As a non-limiting example, the formulation can include a polynucleotide complexed with a polymer and a metal cation (see, for example, U.S. Patent Nos. 6,265,389 and 6,555,525, each of which is incorporated herein by reference in its entirety).

[0409] The nucleic acid molecule (e.g., plasmid, mRNA, DNA) or virus can be formulated as the sole pharmaceutically active ingredient in the pharmaceutical composition, or can be combined with other active agents for the specific disorder to be treated. Optionally, other medicinal agents, pharmaceutical agents, carriers, adjuvants, diluents can be included in the compositions provided herein. For example, any one or more of wetting agents, emulsifying agents, and lubricating agents such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweeteners, flavorings and perfuming agents, preservatives, antioxidants, chelating agents, and inert gases can also be present in the composition. Exemplary other agents and excipients that can be included in the compositions include, for example, water-soluble antioxidants such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, and the like; oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, α-tocopherol, and the like; and metal chelating agents such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, and phosphoric acid, and the like.

[0410] 6.11.How to use The T cell activators of the present disclosure elicit an immune response and are useful for treating or preventing disease states in which stimulation of the host's immune system is beneficial, particularly those in which an enhanced cell-mediated immune response is desirable. These may include disease states in which the host's immune response is insufficient or deficient. Disease states in which the T cell activators of the present disclosure can be administered for treatment or prevention include, for example, tumors or infectious diseases, in which the cell-mediated immune response is an important mechanism for specific immunity. Particular disease states in which the T cell activators of the present disclosure can be used include cancer. The T cell activators of the present disclosure may be administered by themselves or in any suitable pharmaceutical composition, including those described in Section 6.10.1 (e.g., when administered as a T cell activator polypeptide) or Sections 6.10.2 and 6.10.2.1 (e.g., when administered as one or more nucleic acids encoding the T cell activator).

[0411] In one aspect, a T cell activator of the present disclosure is provided for use as a pharmaceutical. In a further aspect, a T cell activator of the present disclosure is provided for use in treating a disease. In certain embodiments, a T cell activator of the present disclosure is provided for use in a method of treatment. In one embodiment, the present disclosure provides a T cell activator described herein for use in treating a disease in a subject in need thereof. In certain embodiments, the present disclosure provides a T cell activator for use in a method of treating a subject having a disease, the method comprising administering to the individual a therapeutically effective amount of the T cell activator. In certain embodiments, the disease being treated is a proliferative disorder. In a preferred embodiment, the disease is cancer. In certain embodiments, when the disease being treated is cancer, the method further comprises administering to the individual a therapeutically effective amount of at least one additional therapeutic agent, e.g., an anti-cancer agent, e.g., a multispecific antigen-binding molecule described in Section 6.6.

[0412] In further embodiments, the present disclosure provides a T cell activator for use in stimulating the immune system, particularly in an antigen-specific manner. In certain embodiments, the present disclosure provides a T cell activator for use in a method of stimulating the immune system in a subject, comprising administering to the individual an effective amount of the T cell activator to stimulate the immune system. The "individual" according to any of the above embodiments is a mammal, preferably a human. "Stimulation of the immune system" according to any of the above embodiments can include any one or more of the following: a general increase in immune function, an increase in T cell function, an increase in T cell proliferation (particularly antigen-specific T cell proliferation), an increase in B cell function, restoration of lymphocyte function, an increase in T cell responsiveness, an increase in natural killer cell activity or lymphokine-activated killer (LAK) cell activity, and the like. In certain embodiments, the present disclosure provides a T cell activator for use in increasing antigen-specific T cell proliferation, wherein the T cell activator is for use in increasing the proliferation of T cells expressing a TCR specific for an antigen (or portion thereof) present in the pMHC complex of the T cell activator.

[0413] In further embodiments, the present disclosure provides methods for preventing a disorder or condition associated with a specific antigen, comprising administering a T cell activator of the present disclosure to an individual. As disclosed herein, the T cell activator may be used to activate T cells in an antigen-specific manner, thereby inducing a protective immune response in the individual. Such methods, also referred to herein as "prophylactic methods," comprise administering a T cell activator of the present disclosure to a subject at risk of developing a disease, disorder, or condition associated with the antigen, wherein the T cell activator comprises a pMHC complex comprising the antigen or a portion thereof. Antigens include viral antigens, bacterial antigens, and tumor antigens, and the prophylactic methods of the present disclosure may include, for example, the prevention of malignant tumors. In some embodiments, the present disclosure provides T cell activators for use in prophylactic methods. Exemplary nucleic acids and formulations for delivery of T cell activators are described in Sections 6.10.2.1 and 6.10.2, respectively.

[0414] In certain embodiments, the prophylactic methods of the present disclosure involve administering a T cell activator comprising a tumor antigen (or a portion thereof), thereby preventing malignant tumors in an individual. Such tumor antigens (or portions thereof; also referred to herein as "cancer cell antigenic determinants" or "antigenic peptides") include peptides gp33-41 from LCMV, APF(126-134), BALF(276-284), CEA(571-579), CMV pp65(495-503), FLU-M1(58-66), gp100(154-162), gp100(209-217), HBV Core(18-27), Her2 / neu(369-377; V2v9); HPV E7(11-20), HPV E7(11-19), HPV Additional tumor antigens contemplated herein include, but are not limited to, E7(82-90), KLK4(11-19), LMP1(125-133), MAG-A3(112-120), MAGE-A4(230-239), MAGE-A4(286-294), NYESO1(157-165, C165A), NYESO1(157-165, C165V), p54 WT(264-272), PAP-3(136-143), PSMA(4-12), PSMA(135-145), survivin(96-014), tyrosinase(369-377, 371D), and WT1(126-134). Additional tumor antigens contemplated herein include those listed in Table 2. An "individual" according to any of the above embodiments is a mammal, preferably a human.

[0415] In a further aspect, the present disclosure provides use of a T cell activator of the present disclosure in the manufacture or preparation of a medicament for treating or preventing a disease in a subject in need thereof. In one embodiment, the medicament is for use in a method of treating a disease, comprising administering a therapeutically effective amount of the medicament to a subject having the disease. In certain embodiments, the disease being treated is a proliferative disorder. In a preferred embodiment, the disease is cancer. In one such embodiment, the method further comprises, when the disease being treated is cancer, administering to the individual a therapeutically effective amount of at least one additional therapeutic agent, e.g., an anti-cancer agent, e.g., a multispecific antigen-binding molecule described in Section 6.6. In a further embodiment, the medicament is for use in a method of preventing a disease, comprising administering a prophylactically effective amount of the medicament to a subject not having the disease, optionally a subject at risk of developing the disease. In certain embodiments, the disease being prevented is a proliferative disorder.

[0416] In a further embodiment, the medicament is for stimulating the immune system. In a further embodiment, the medicament is for use in a method of stimulating the immune system in a subject, the method comprising administering to the individual an amount of the medicament effective to stimulate the immune system. The "individual" according to any of the above embodiments is a mammal, preferably a human. "Stimulation of the immune system" according to any of the above embodiments may include any one or more of a general increase in immune function, an increase in T cell function, an increase in T cell proliferation (particularly antigen-specific T cell proliferation), an increase in B cell function, restoration of lymphocyte function, an increase in T cell responsiveness, an increase in natural killer cell activity or lymphokine-activated killer (LAK) cell activity, and the like.

[0417] In a further aspect, the present disclosure provides a method for treating a disease in a subject, comprising administering to the individual a therapeutically effective amount of a T cell activator of the present disclosure. In one embodiment, a composition comprising a T cell activator of the present disclosure in a pharmaceutically acceptable form is administered to the individual. In certain embodiments, the disease being treated is a proliferative disorder. In a preferred embodiment, the disease is cancer. In certain embodiments, when the disease being treated is cancer, the method further comprises administering to the individual a therapeutically effective amount of at least one additional therapeutic agent, e.g., an anti-cancer agent, e.g., a multispecific antigen-binding molecule described in Section 6.6. In a further aspect, the present disclosure provides a method for stimulating the immune system in a subject, comprising administering to the individual an effective amount of a T cell activator to stimulate the immune system. An "individual" according to any of the above embodiments is a mammal, preferably a human.

[0418] In certain embodiments, the disease to be treated is a proliferative disorder, preferably cancer.Non-limiting examples of cancer include bladder cancer, brain cancer, head and neck cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, uterine cancer, cervical cancer, endometrial cancer, esophageal cancer, colon cancer, colorectal cancer, rectal cancer, gastric cancer, prostate cancer, blood cancer, skin cancer, squamous cell carcinoma, bone cancer, and kidney cancer.Other cell proliferative disorders that can be treated using the T cell activator of the present disclosure include, but are not limited to, neoplasms located in the abdomen, bone, breast, digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal glands, parathyroid glands, pituitary gland, testes, ovaries, thymus, thyroid gland), eye, head and neck, nervous system (central and peripheral), lymphatic system, pelvic system, skin, soft tissue, spleen, chest region, and genitourinary system.Also included are precancerous conditions or lesions and cancer metastasis. In certain embodiments, the cancer is selected from the group consisting of renal cell carcinoma, skin cancer, lung cancer, colorectal cancer, breast cancer, brain cancer, and head and neck cancer. Similarly, other cell proliferative disorders can be treated with the T cell activators of the present disclosure. Examples of such cell proliferative disorders include, but are not limited to, hypergammaglobulinemia, lymphoproliferative disorders, dysproteinemia, purpura, sarcoidosis, Sézary syndrome, Waldenstrom's macroglobulinemia, Gaucher disease, histiocytosis, and any other cell proliferative disease other than neoplasms located in the above-mentioned organ systems. Those skilled in the art will readily recognize that in many cases, when used in the context of therapeutic treatment, T cell activators may not provide a cure but may only provide partial benefit. In some embodiments, physiological changes that have some benefit are also considered therapeutically beneficial. Thus, in some embodiments, the amount of T cell activator that produces a physiological change is considered an "effective amount" or a "therapeutically effective amount." Those skilled in the art will further recognize that, in many cases, when used in the context of prophylactic treatment, T cell activators may not provide a cure, but may prevent a subject from acquiring a particular disease or disorder altogether, but may reduce the likelihood of developing the disease or disorder. The subject, patient, or individual in need of treatment is typically a mammal, more particularly a human.

[0419] The appropriate dosage of the T cell activator of the present disclosure (when used alone or in combination with one or more other additional therapeutic agents) for the prevention or treatment of disease will depend on the type of disease being treated, the route of administration, the patient's weight, the specific T cell activator, the severity and course of the disease, whether the T cell activator is administered for prophylactic or therapeutic purposes, previous or concurrent therapeutic interventions, the patient's clinical history and response to the T cell activator, and the judgment of the attending physician. In any event, the medical professional responsible for administration will determine the concentration of active ingredient(s) in the composition and the appropriate dose(s) for the individual subject. Various administration schedules are contemplated herein, including, but not limited to, single or multiple administrations over various time points, bolus administration, and pulse infusion.

[0420] The T cell activator of the present disclosure will generally be used in an amount effective to achieve its intended purpose. For use in treating or preventing a disease state, the T cell activator of the present disclosure or a pharmaceutical composition thereof will be administered or applied in a therapeutically effective amount. Determining a therapeutically effective amount is well within the capabilities of those skilled in the art, especially in light of the detailed disclosure provided herein.

[0421] For systemic administration, the therapeutically effective dose can be estimated initially from in vitro assays, such as cell culture assays. The EC 50 A dose can be formulated in animal models to achieve a circulating concentration range including, but not limited to, 100 mg / kg / day. Such information can be used to more accurately determine useful doses in humans.

[0422] Initial doses can also be estimated from in vivo data, such as animal models, using techniques known in the art. Those skilled in the art would be readily able to optimize human administration based on animal data.

[0423] Dosage and interval can be individually adjusted to provide plasma levels of T cell activator sufficient to maintain therapeutic efficacy. Typical patient dosages for administration by injection range from about 0.1 to 50 mg / kg / day, typically about 0.5 to 1 mg / kg / day. Therapeutically effective plasma levels can be achieved by multiple daily administrations. Plasma levels can be measured, for example, by ELISA or HPLC.

[0424] In cases of local administration or selective uptake, the effective local concentration of T cell activator may not be related to plasma concentration. One skilled in the art will be able to optimize the therapeutically effective local dosage without undue experimentation.

[0425] The therapeutically effective dose of the T cell activators described herein will generally provide therapeutic benefit without causing substantial toxicity. The toxicity and therapeutic efficacy of T cell activators can be determined by standard pharmaceutical procedures in cell cultures or experimental animals. Using cell culture assays and animal studies, LD 50 (the dose that is lethal to 50% of the population) and ED 50 The dose ratio between toxic and therapeutic effects is the therapeutic index, which is the LD 50 / ED 50 Therapeutic indices can be expressed as a ratio of ED . T cell activators that exhibit large therapeutic indices are preferred. In one embodiment, the T cell activators according to the present disclosure exhibit a high therapeutic index. Data obtained from cell culture assays and animal studies can be used in formulating a range of dosages suitable for use in humans. Dosages are preferably within the ED range with little or no toxicity. 50The circulating concentration range includes the range of 100-200 mg / kg / day. The dosage can vary within this range depending on various factors, such as the dosage form used, the route of administration utilized, the condition of the subject, etc. The exact formulation, route of administration, and dosage can be selected by the individual physician in consideration of the patient's condition. (See, for example, Fingl et al., 1975, In: The Pharmacological Basis of Therapeutics, Ch. 1, p. 1, which is incorporated herein by reference in its entirety.)

[0426] The attending physician of a patient treated with a T cell activator of the present disclosure will know how and when to terminate, interrupt, or adjust administration due to toxicity, organ dysfunction, etc. Conversely, the attending physician will also know how to adjust treatment to higher levels if the clinical response is not adequate (excluding toxicity). The magnitude of an administered dose in the management of the disorder of interest will vary depending on the severity of the condition being treated, the route of administration, and the like. The severity of the condition may, for example, be assessed, in part, by standard prognostic evaluation methods. Furthermore, the dose, and perhaps the frequency of administration, will also vary according to the age, weight, and response of the individual patient.

[0427] 6.12. Combination Therapy T cell activators according to the present disclosure may be administered in combination with one or more other agents in a treatment. For example, T cell activators of the present disclosure may be co-administered with at least one additional therapeutic agent. The term "therapeutic agent" encompasses any agent administered to treat a condition or disease in a subject in need of such treatment. Such additional therapeutic agents may include any active ingredient suitable for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. In certain embodiments, the additional therapeutic agent is an immunomodulatory agent, a cytostatic agent, a cell adhesion inhibitor, a cytotoxic agent, an activator of cell apoptosis, or an agent that increases the sensitivity of cells to apoptosis inducers. In certain embodiments, the additional therapeutic agent is an anti-cancer agent, such as a microtubule-disrupting agent, an antimetabolite, a topoisomerase inhibitor, a DNA intercalator, an alkylating agent, a hormone therapy agent, a kinase inhibitor, a receptor antagonist, an activator of tumor cell apoptosis, or an anti-angiogenic agent. In certain embodiments, the additional therapeutic agent is a multispecific antigen-binding molecule described in Section 6.6, including, but not limited to, the multispecific antigen-binding molecules of Table K-2.

[0428] Such active ingredients are preferably present in combination in amounts effective for the intended purpose. The effective amount of such other agents will depend on the amount of T cell activator used, the type of disorder or treatment, and other factors discussed above. T cell activators are generally used in the same dosages and by any route of administration described herein, or at about 1 to 99% of the dosages described herein, or at any dosage and by any route determined empirically / clinically appropriate.

[0429] Such combination therapies as described above encompass combined administration (wherein two or more therapeutic agents are contained in the same or separate compositions) and separate administration, where administration of the T cell activators of the present disclosure can occur before, simultaneously with, and / or after administration of the additional therapeutic agent(s) and / or adjuvant. The T cell activators of the present disclosure can also be used in combination with radiation therapy.

[0430] 7. Specific Embodiments While various specific embodiments have been illustrated and described, it will be understood that various changes can be made without departing from the spirit and scope of the present disclosure(s), which is exemplified by the numbered embodiments set forth below.

[0431] In certain aspects of the following numbered embodiments and claims below, the Fc domain, MHC domain, β2M, and variants thereof preferably comprise the amino acid sequence of a human Fc domain, human MHC domain, human β2M, and variants thereof, e.g., variants having at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to such human sequences.

[0432] 1. (a) a peptide-MHC (pMHC) complex comprising an MHC domain and an antigenic peptide; (b) an immune cell antigen (ICA) targeting moiety; and (c) o...

Claims

1. (a) a peptide-MHC (pMHC) complex comprising an MHC domain and an antigenic peptide; (b) an immune cell antigen (ICA) targeting moiety; and (c) optionally a multimerization moiety operably linked to said pMHC complex.

2. The multispecific molecule of claim 1 , comprising a single ICA targeting moiety.

3. The multispecific molecule of claim 1 , comprising two ICA targeting moieties.

4. 4. The multispecific molecule of claim 3, wherein the two ICA targeting moieties bind to the same antigen, and optionally the two ICA targeting moieties are the same.

5. The multispecific molecule of claim 3 , wherein the two ICA targeting moieties bind to different antigens.

6. 6. The multispecific molecule of any one of claims 1 to 5, comprising a single pMHC complex.

7. 6. The multispecific molecule of claim 1, comprising two pMHC complexes.

8. 8. The multispecific molecule of claim 7, wherein the two pMHC complexes are the same.

9. 9. The multispecific molecule of claim 1, comprising a multimerization moiety operably linked to said pMHC complex, said multimerization moiety being an Fc domain.

10. The multispecific molecule of any one of claims 1 to 9, wherein the multispecific molecule is a heterodimer.

11. The multispecific molecule of any one of claims 2 to 10, wherein the pMHC complex is N-terminal to the multimerization moiety.

12. The multispecific molecule of any one of claims 2 to 10, wherein the pMHC complex is C-terminal to the multimerization moiety.

13. The multispecific molecule of any one of claims 2 to 12, wherein the ICA targeting moiety is N-terminal to the multimerization moiety.

14. The multispecific molecule of any one of claims 2 to 12, wherein the ICA targeting moiety is C-terminal to the multimerization moiety.

15. The multispecific molecule of any one of claims 1 to 14, wherein the pMHC complex and the ICA targeting moiety are on a single polypeptide.

16. The multispecific molecule of any one of claims 1 to 14, wherein the pMHC complex and the ICA targeting moiety are on different polypeptides.

17. The multispecific molecule of any one of claims 1 to 16, wherein the ICA targeting moiety is a T cell antigen (TCA) targeting moiety.

18. 18. The multispecific molecule of claim 17, wherein the TCA targeting moiety is a CD3 targeting moiety.

19. 18. The multispecific molecule of claim 17, wherein the TCA targeting moiety is a CD28 targeting moiety.

20. The multispecific molecule of any one of claims 1 to 16, wherein the ICA targeting moiety is a B cell antigen (BCA) targeting moiety.

21. 21. The multispecific molecule of claim 20, which lacks a TCA targeting moiety.

22. 22. The multispecific molecule of claim 20 or 21, wherein the BCA targeting moiety is a CD19 targeting moiety.

23. 22. The multispecific molecule of claim 20 or 21, wherein the BCA targeting moiety is a CD20 targeting moiety.

24. 22. The multispecific molecule of claim 20 or 21, wherein the BCA targeting moiety is a CD22 targeting moiety.

25. The multispecific molecule of any one of claims 1 to 24, further comprising a tumor antigen targeting moiety.

26. 26. The multispecific molecule of claim 25, wherein the tumor antigen targeting moiety is a tumor-associated antigen (TAA) targeting moiety.

27. The multispecific molecule of any one of claims 1 to 26, wherein the MHC domain is an MHC class I domain.

28. 28. The multispecific molecule of claim 27, wherein the pMHC complex further comprises a β2-microglobulin domain.

29. The multispecific molecule of any one of claims 1 to 28, wherein the pMHC complex is a single polypeptide.

30. 30. The multispecific molecule of any one of claims 1 to 29, wherein the multispecific molecule comprises a polypeptide comprising, in N-terminal to C-terminal orientation, the pMHC complex and an Fc domain.

31. (a) (i) a peptide-MHC (pMHC) complex comprising an MHC domain and an antigenic peptide; (ii) a first polypeptide chain comprising a first Fc domain; (b)(i) VH; and (ii) a second polypeptide chain comprising a second Fc domain that associates with the first Fc domain to form an Fc region; (c) (i) a third polypeptide comprising a VL that associates with said VH to form an immune cell antigen (ICA) targeting moiety.

32. 32. The multispecific molecule of claim 31 , wherein the pMHC complex is N-terminal to the first Fc domain.

33. 32. The multispecific molecule of claim 31 , wherein the pMHC complex is C-terminal to the first Fc domain.

34. The multispecific molecule of any one of claims 31 to 33, wherein the VH is N-terminal to the second Fc domain.

35. The multispecific molecule of any one of claims 31 to 33, wherein the VH is C-terminal to the second Fc domain.

36. The multispecific molecule of any one of claims 31 to 35, wherein the ICA targeting moiety is a T cell antigen (TCA) targeting moiety.

37. 37. The multispecific molecule of claim 36, wherein the TCA targeting moiety is a CD3 targeting moiety.

38. 37. The multispecific molecule of claim 36, wherein the TCA targeting moiety is a CD28 targeting moiety.

39. 36. The multispecific molecule of any one of claims 31 to 35, wherein the ICA targeting moiety is a B cell antigen (BCA) targeting moiety.

40. 40. The multispecific molecule of claim 39, which lacks a TCA targeting moiety.

41. 41. The multispecific molecule of claim 39 or 40, wherein the BCA targeting moiety is a CD19 targeting moiety.

42. 41. The multispecific molecule of claim 39 or 40, wherein the BCA targeting moiety is a CD20 targeting moiety.

43. 41. The multispecific molecule of claim 39 or 40, wherein the BCA targeting moiety is a CD22 targeting moiety.

44. 44. The multispecific molecule of any one of claims 31 to 43, further comprising a tumor antigen targeting moiety.

45. 45. The multispecific molecule of claim 44, wherein the tumor antigen targeting moiety is a tumor-associated antigen (TAA) targeting moiety.

46. 46. ​​The multispecific molecule of any one of claims 31 to 45, wherein the MHC domain is an MHC class I domain.

47. The multispecific molecule of any one of claims 31 to 46, wherein the pMHC complex further comprises a β2-microglobulin domain.

48. The multispecific molecule of any one of claims 1 to 47, wherein the antigenic peptide is an antigenic peptide listed in Table 2-C.

49. A nucleic acid or nucleic acids encoding the multispecific molecule of any one of claims 1 to 48.

50. 100. A cell transfected with one or more expression vectors comprising one or more nucleic acid sequences encoding the multispecific molecule of any one of claims 1 to 48 under the control of one or more promoters.

51. 51. A method of producing a multispecific molecule according to any one of claims 1 to 48, comprising culturing a host cell according to claim 50 and recovering the multispecific molecule expressed thereby.

52. 50. A method of activating T cell receptor signaling in a T cell or a population of T cells, comprising administering to said T cell or population of T cells a multispecific molecule of any one of claims 1 to 48.

53. 100. A pharmaceutical composition comprising (a) a multispecific molecule of any one of claims 1 to 48, or one or more nucleic acids encoding the multispecific molecule of any one of claims 1 to 48, and (b) an excipient.

54. 50. A method of treating cancer comprising administering to a subject in need thereof, wherein the multispecific molecule is the multispecific molecule of any one of claims 1 to 48 or the nucleic acid(s) of claim 49.

55. 50. A method of inhibiting the growth of tumor cells in a subject, comprising administering to the subject a multispecific molecule which is the multispecific molecule of any one of claims 1 to 48 or the nucleic acid(s) of claim 49.

56. 50. A method of stimulating the proliferation of cancer antigen-specific T cells, comprising administering to a subject, wherein the multispecific molecule is a multispecific molecule of any one of claims 1 to 48 or a nucleic acid(s) of claim 49.

57. 57. The method of any one of claims 54 to 56, wherein administration of the multispecific molecule reduces the viability of cancer cells in the subject that express a protein comprising the amino acid sequence of the antigenic peptide.

58. 57. The method of any one of claims 54 to 56, further comprising administering to the subject a multispecific antigen-binding molecule comprising: (a) a first antigen-binding domain that specifically binds to a tumor antigen; and (b) a second antigen-binding domain that specifically binds to a T-cell antigen.

59. 59. The method of claim 58, wherein the second antigen-binding domain is a CD28-binding domain.

60. 50. A method of stimulating the proliferation of antigen-specific T cells, comprising administering to a subject, wherein the multispecific molecule is a multispecific molecule of any one of claims 1 to 48 or a nucleic acid(s) of claim 49.

61. 54. A method comprising administering to a subject a multispecific molecule of any one of claims 1 to 48, one or more nucleic acids encoding the multispecific molecule of any one of claims 1 to 48, or a pharmaceutical composition of claim 53.

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