Recombinant Fc Domain-IL2 variant polypeptides and combination therapies with membrane anchor antigen binding polypeptides

Abstract

The present invention relates to a combination therapy of a recombinant Fc domain-IL-2 variant polypeptide with a membrane anchor antigen binding polypeptide in the prevention or treatment of cancer.

Description

Technical Field

[0001] The present invention relates to a combination therapy of a recombinant Fc domain-IL-2 variant polypeptide and a membrane-anchored antigen-binding polypeptide for the prevention or treatment of cancer. Background Art

[0002] Adoptive cell therapy has become a clinically validated cancer treatment. Although chimeric antigen receptor (CAR)-T cell therapy has shown clinical efficacy in hematological malignancies, several obstacles still need to be overcome in order to be effective in solid tumors. A key challenge of CAR-T therapy for solid tumors is on-target off-tumor toxicity, which is triggered by the limited but significant expression of tumor antigens in healthy tissues. As a result, systemic cytokine release induced by CAR-T cells is often observed and may lead to various treatment-related symptoms (including neurotoxicity).

[0003] Adaptor-based CARs include tumor antigen-specific adaptor molecules and CARs with unique specificity for the adaptor molecules. The corresponding adaptor CAR-T cells can only be activated in the presence of adaptor molecules bound to antigen-positive cells, which enables control of their therapeutic activity and systemic toxicity. The present inventors have previously reported a modular adaptor CAR-T approach that uses a recombinant antibody with a mutant effector function-silencing Fc domain as an adaptor molecule (Darowski et al. (2019), and disclosed in WO 2018 / 177966 A1). The homologous CAR expressed by the engineered T cells is specific for the above-mentioned mutant Fc variant containing the previously described P329G mutation. Importantly, in many clinical trials, recombinant antibody-based biologics with Fc variants combining the P329G mutation with the L234AL235A mutation were shown to be essentially Fc effector function-silent and non-immunogenic.

[0004] In addition to toxicity, another major obstacle to solid tumor CAR-T therapy is the exhaustion and limited persistence of engineered T cells in patients. For current autologous CAR-T cell therapy, cells are collected from patients and the cells are engineered and amplified in vitro with a cytokine mixture (usually containing interleukin (IL) -2, but also containing IL-7, IL-15 and / or IL-21) (Zhang et al., 2020). This amplification step is necessary to achieve high cell counts, but it also leads to terminal T cell differentiation and exhaustion, resulting in limited persistence and lower effectiveness. In addition, such T cell products are typically a mixture of non-engineered cells and engineered cells, so that the cell composition in the final product is not optimal. There is a trend to shorten the ex vivo amplification process in order to limit cell differentiation and push cells more towards stem cell-like memory T cell phenotypes. Shortening the ex vivo amplification time and amplifying T cells directly in the patient's body will have several benefits, including saving production costs and time and reducing process-mediated differentiation of T cells. However, to date, a clinically validated program for enabling CAR-T cells to specifically amplify in patients has not yet been established.

[0005] Conventional IL-2 therapy has dose-limiting systemic side effects mediated by, for example, regulatory T cells or endothelial cells. To overcome this limitation, researchers have developed novel IL-2-based therapies. For example, it has been shown that cis-targeting IL-2 to the desired tumor-reactive T cell population has significantly improved toxicity characteristics. Different strategies for cis-targeting have been reported, including, for example, CD8 targeting (Sultan et al. (2021)) or PD1 targeting (Deak et al. (2022)). Summary of the Invention

[0006] The present invention comprises a combination therapy of a recombinant Fc domain-IL2 variant (Fc-IL2v) polypeptide complex and a membrane-anchored antigen binding (MAB) polypeptide or MAB polypeptide complex for use as a combination therapy, particularly in the treatment of cancer.

[0007] for use as combination therapy in the prevention or treatment of metastasis, or

[0008] For use as combination therapy in stimulating immune responses or functions such as T cell activity.

[0009] In one aspect, a recombinant Fc domain-IL2 variant (Fc-IL2v) polypeptide complex in combination with a recombinant membrane-anchored antigen binding (MAB) polypeptide or MAB polypeptide complex is provided for use in treating cancer, in preventing or treating metastasis, or in stimulating an immune response or function such as T cell activity.

[0010] The recombinant Fc-IL2v polypeptide complex comprises:

[0011] (i) a first polypeptide comprising a variant CH2-CH3 region comprising G329 according to EU numbering, and wherein the first polypeptide further comprises an IL-2 variant (IL2v) polypeptide comprising an IL-2 polypeptide comprising amino acid substitutions F42A, Y45A, and L72G, wherein the numbering is relative to the human IL-2 sequence SEQ ID NO: 40; and

[0012] (ii) a second polypeptide comprising a variant CH2-CH3 region comprising G329 according to EU numbering, wherein the recombinant Fc-IL2v polypeptide complex does not comprise an antigen binding portion, and

[0013] wherein the MAB polypeptide or MAB polypeptide complex comprises an antigen binding portion or a component thereof, and a transmembrane domain, wherein the antigen binding portion binds to a variant CH2-CH3 region comprising an amino acid substitution P329G according to EU numbering, relative to the amino acid sequence of a reference CH2-CH3 region comprising P329 according to EU numbering, to which the antigen binding portion does not bind.

[0014] In one aspect, a recombinant Fc domain-IL2 variant (Fc-IL2v) polypeptide complex is provided, comprising:

[0015] (i) a first polypeptide comprising a variant CH2-CH3 region comprising G329 according to EU numbering, and wherein the first polypeptide further comprises an IL-2 variant (IL2v) polypeptide comprising an IL-2 polypeptide comprising amino acid substitutions F42A, Y45A, and L72G, wherein the numbering is relative to the human IL-2 sequence SEQ ID NO: 40; and

[0016] (ii) a second polypeptide comprising a variant CH2-CH3 region comprising G329 according to EU numbering, wherein the recombinant Fc-IL2v polypeptide complex does not comprise an antigen binding portion.

[0017] In one aspect, a method for treating or preventing cancer in an individual or for stimulating an immune response or function such as T cell activity in an individual is provided, wherein the method comprises

[0018] (a) administering to the individual a recombinant Fc domain-IL2 variant (Fc-IL2v) polypeptide complex, wherein the recombinant Fc-IL2v polypeptide complex comprises:

[0019] (i) a first polypeptide comprising a variant CH2-CH3 region comprising G329 according to EU numbering, and wherein the first polypeptide further comprises an IL-2 variant (IL2v) polypeptide comprising an IL-2 polypeptide comprising amino acid substitutions F42A, Y45A, and L72G, wherein the numbering is relative to the human IL-2 sequence SEQ ID NO: 40; and

[0020] (ii) a second polypeptide comprising a variant CH2-CH3 region comprising G329 according to EU numbering; and

[0021] (b) administering a recombinant membrane-anchored antigen binding (MAB) polypeptide or MAB polypeptide complex, wherein the MAB polypeptide or MAB polypeptide complex comprises an antigen binding portion, or a component thereof, and a transmembrane domain, wherein the antigen binding portion binds to a variant CH2-CH3 region comprising an amino acid substitution P329G according to EU numbering, relative to the amino acid sequence of a reference CH2-CH3 region comprising P329 according to EU numbering, to which the antigen binding portion does not bind.

[0022] In one aspect, there is provided a use of a recombinant Fc domain-IL2 variant (Fc-IL2v) polypeptide complex in the manufacture of a medicament for treating or preventing cancer in an individual, or for stimulating an immune response or function, such as T cell activity, in an individual, wherein the recombinant Fc-IL2v polypeptide complex comprises:

[0023] (i) a first polypeptide comprising a variant CH2-CH3 region comprising G329 according to EU numbering, and wherein the first polypeptide further comprises an IL-2 variant (IL2v) polypeptide comprising an IL-2 polypeptide comprising amino acid substitutions F42A, Y45A, and L72G, wherein the numbering is relative to the human IL-2 sequence SEQ ID NO: 40; and

[0024] (ii) a second polypeptide comprising a variant CH2-CH3 region comprising G329 according to EU numbering.

[0025] In one aspect, there is provided use of a recombinant Fc domain-IL2 variant (Fc-IL2v) polypeptide complex in the manufacture of a medicament for treating or preventing cancer in an individual, or for stimulating an immune response or function, such as T cell activity, in an individual, wherein the treatment comprises:

[0026] (a) administering to the individual a recombinant Fc domain-IL2 variant (Fc-IL2v) polypeptide complex, wherein the recombinant Fc-IL2v polypeptide complex comprises:

[0027] (i) a first polypeptide comprising a variant CH2-CH3 region comprising G329 according to EU numbering, and wherein the first polypeptide further comprises an IL-2 variant (IL2v) polypeptide comprising an IL-2 polypeptide comprising amino acid substitutions F42A, Y45A, and L72G, wherein the numbering is relative to the human IL-2 sequence SEQ ID NO: 40; and

[0028] (ii) a second polypeptide comprising a variant CH2-CH3 region comprising G329 according to EU numbering; and

[0029] (b) administering a recombinant membrane-anchored antigen binding (MAB) polypeptide or MAB polypeptide complex, wherein the MAB polypeptide or MAB polypeptide complex comprises an antigen binding portion, or a component thereof, and a transmembrane domain, wherein the antigen binding portion binds to a variant CH2-CH3 region comprising an amino acid substitution P329G according to EU numbering, relative to the amino acid sequence of a reference CH2-CH3 region comprising P329 according to EU numbering, to which the antigen binding portion does not bind.

[0030] In some aspects, the antigen binding portion that binds to Fc-IL2v comprises the heavy chain variable (VH) and light chain variable (VL) regions of an antibody bound to a variant CH2-CH3 region comprising the amino acid substitution P329G according to EU numbering.

[0031] In some aspects, the antigen binding portion is or comprises an Fv, scFv, Fab, Fab', Fab'-SH, F(ab')2, crossFab, scFab, or dAb portion.

[0032] In one aspect, the antigen binding portion comprises:

[0033] (a)(i) a VH region comprising the following CDRs:

[0034] an HC-CDR1 having the amino acid sequence of SEQ ID NO: 11;

[0035] an HC-CDR2 having the amino acid sequence of SEQ ID NO: 19; and

[0036] an HC-CDR3 having the amino acid sequence of SEQ ID NO: 13;

[0037] as well as

[0038] (ii) a VL region comprising the following CDRs:

[0039] LC-CDR1 having the amino acid sequence of SEQ ID NO: 24;

[0040] LC-CDR2 having the amino acid sequence of SEQ ID NO: 25; and

[0041] an LC-CDR3 having the amino acid sequence of SEQ ID NO: 26;

[0042] or

[0043] (b)(i) a VH region comprising the following CDRs:

[0044] an HC-CDR1 having the amino acid sequence of SEQ ID NO: 11;

[0045] an HC-CDR2 having the amino acid sequence of SEQ ID NO: 12; and

[0046] an HC-CDR3 having the amino acid sequence of SEQ ID NO: 13;

[0047] as well as

[0048] (ii) a VL region comprising the following CDRs:

[0049] LC-CDR1 having the amino acid sequence of SEQ ID NO: 24;

[0050] LC-CDR2 having the amino acid sequence of SEQ ID NO: 25; and

[0051] LC-CDR3 having the amino acid sequence of SEQ ID NO: 26.

[0052] In some aspects, the mutant IL-2 polypeptide further comprises the amino acid substitution Q126T.

[0053] In some aspects, the recombinant Fc-IL2v polypeptide complex does not comprise an antigen binding portion, particularly wherein the recombinant Fc-IL2v polypeptide complex does not comprise a scFv, Fab, or crossFab.

[0054] In some aspects, the recombinant MAB polypeptide comprises an amino acid sequence derived from IL2Ra, IL15Ra, or CD8a.

[0055] In some aspects, the recombinant MAP polypeptide is a chimeric antigen receptor (CAR).

[0056] In some aspects, the recombinant MAP polypeptide comprises at least one recombinant CD3-TCR complex polypeptide.

[0057] In some aspects, the recombinant CD3-TCR complex polypeptide comprises:

[0058] (i) an antigen binding portion or component thereof, wherein the antigen binding portion binds to a variant CH2-CH3 region comprising an amino acid substitution, P329G, according to EU numbering, relative to the amino acid sequence of a reference CH2-CH3 region comprising P329 according to EU numbering, to which the antigen binding portion does not bind; and

[0059] (ii) a CD3-TCR complex association domain having an amino acid sequence derived from a CD3-TCR complex polypeptide.

[0060] In some aspects, the recombinant CD3-TCR complex polypeptide is capable of associating with one or more CD3-TCR complex polypeptides via its CD3-TCR complex association domain to form a CD3-TCR complex.

[0061] In some aspects, the amino acid sequence derived from a CD3-TCR complex polypeptide is derived from CD3ε, TCRα, or TCRβ.

[0062] In some aspects, a cell comprising a recombinant MAB polypeptide or MAB polypeptide complex as described above is provided.

[0063] In some aspects, a method for producing an enriched cell pool is provided, the method comprising: contacting a starting cell pool comprising at least one cell as described above with a recombinant Fc-IL2v polypeptide complex as described above; and incubating the cells until the proportion of cells comprising the recombinant MAB polypeptide or the MAB polypeptide complex reaches a desired proportion of the total cell pool to produce an enriched cell pool.

[0064] In some aspects, a nucleic acid or multiple nucleic acids are provided, which encode a recombinant Fc domain-IL2 variant (Fc-IL2v) polypeptide complex as described above or a recombinant MAB polypeptide or MAB polypeptide complex as described above.

[0065] In some aspects, an expression vector or multiple expression vectors are provided, comprising a nucleic acid as described above.

[0066] In some aspects, a cell is provided, comprising: a recombinant MAB polypeptide or MAB polypeptide complex as described above; a nucleic acid or nucleic acids as described above; or an expression vector or expression vectors as described above.

[0067] In some aspects, cells expressing recombinant MAB polypeptides and / or recombinant MAB polypeptide complexes are specifically expanded, particularly wherein the cells are specifically expanded by contacting the cells with a recombinant Fc-IL2v polypeptide complex as described above.

[0068] In some aspects, cells expressing recombinant MAB polypeptides and / or recombinant MAB polypeptide complexes are enriched, particularly wherein the cells are enriched by contacting the cells with a recombinant Fc-IL2v polypeptide complex as described above.

[0069] In some aspects, cells expressing the recombinant MAB polypeptide and / or recombinant MAB polypeptide complex are enriched to >90% of the total cell pool.

[0070] In some aspects, a method for producing an enriched cell pool is provided, the method comprising: contacting a starting cell pool comprising at least one cell as described above with a recombinant Fc-IL2v polypeptide complex as described above; and incubating the cells until the proportion of cells comprising the recombinant MAB polypeptide or the MAB polypeptide complex reaches a desired proportion of the total cell pool to produce an enriched cell pool.

[0071] In some aspects, a pharmaceutical composition is provided, comprising: the recombinant Fc domain-IL2 variant (Fc-IL2v) polypeptide complex as described above; the cell as described above; or the enriched cell pool produced as described above.

[0072] In some aspects, there is provided the invention as hereinbefore described with reference to the figures and examples. BRIEF DESCRIPTION OF THE DRAWINGS

[0073] Figures 1A to 1D Schematic diagram of exemplary recombinant membrane-anchored antigen binding (MAB) polypeptides and MAB polypeptide complexes according to the present disclosure. Depicted (from left to right) are recombinant CD3ε-TCR complex polypeptides in a TCR complex, MAB polypeptide complexes based on recombinant TCRαβ, recombinant first-generation or second-generation CARs, and recombinant non-signaling tags (1A) with binding moieties in the form of scFvs. Schematic diagram of gene constructs corresponding to P329G-CAR (1B), TCR-based MAB polypeptide complexes (P329G-CD3ε and P329G-Cαβ) (1C), and non-signaling MAB polypeptides (P329G-tag, 1D).

[0074] Figure 2 Schematic diagram of an exemplary recombinant Fc-IL2v polypeptide complex according to the present disclosure. IL2 variants are fused to variant Fc containing orthogonal mutations via a linker to obtain orthogonal ligands for MAB polypeptides or MAB polypeptide complexes according to the present disclosure.

[0075] Figure 3 Schematic diagram of exemplary recombinant Fc-IL2v polypeptide complexes according to the present disclosure and used in the Examples. Fc_P329G_LALA conjugated IL2v(Q126T) and PD1-IL2v(Q126T) (also containing the P329G_LALA mutation) were used as orthogonal ligands, while Fc_WT or Fc_LALA conjugated molecules were used as non-targeting controls.

[0076] Figures 4A to 4C .Schematic diagram of P329G-CAR and P329G-CD3ε / Cαβ constructs. Figure 4A A second generation chimeric antigen receptor (CAR) with an anti-P329G binding portion in scFv format is depicted. Figure 4B and 4C P329G-CD3ε / P329G-Cαβ constructs are shown in the context of endogenous TCR complexes. Anti-P329G scFv is fused to the CD3ε chain (4B, P329G-CD3εTCR complex), or VH is fused to the CαTCR domain and VL is fused to the CβTCR domain (4C, P329G-CαβTCR complex). The P329G-Cαβ construct can be further stabilized by introducing an interchain disulfide bond between the Cα extracellular domain and the Cβ extracellular domain.

[0077] Figure 5A and 5B Jurkat NFAT (TCR / CD3 effector cells (NFAT), Promega, #J1601) wild type (wt) or Jurkat NFAT after CRISPR-Cas9 knockout of endogenous CD3ε was stained using anti-CD3ε-FITC (1:50, Biolegend, #300406). Figure 6A Shown are stainings following knockdown, with Jurkat NFAT wild-type cells serving as a control. Figure 6B The cell populations before and after sorting for CD3ε-negative cells are shown, and the CD3ε-negative cell population accounts for 99.7%.

[0078] Figure 6A and 6B eGFP expression in Jurkat NFAT CD3εKO cells after lentiviral transduction of P329G-CD3ε (6A) or P329G-CAR (6B) and pool sorting for live, eGFP-positive cells. Mock-transduced cells (cells transduced with empty virus-like particles (VLPs)) served as negative controls.

[0079] Figures 7A to 7C: Surface expression of P329G-CAR or P329G-CD3εTCR in Jurkat NFAT CD3εKO cells (sorted pool) was confirmed by staining with AF647-labeled Fc-P329G LALA, as shown Figure 7A (1), the corresponding staining histogram is depicted in Figure 7B (1). The cells were stained with anti-TCRαβ-BV421 (1:50, Biolegend, #306722) and anti-CD3ε-PE (1:50, Biolegend, #300408) antibodies ( Figure 7A (2,3)) staining to assess its integration into the TCR complex and its expression on the cell surface. Figure 7B (2, 3) and Figure 7C (2, 3) Mock-transduced cells (light grey) were used as a negative control for staining.

[0080] Figure 8A and 8B In the presence of FolR1 + In the case of target cells, the activation of Jurkat NFAT CD3εKO cells transduced with P329G-CAR (sorting pool) or P329G-CD3ε (sorting pool), the target cells have high target expression levels (HeLa) or low target expression levels (HT-29) after stimulating with anti-FolR1 (clone 16D5) IgG containing the P329G LALA mutation. Activation was assessed by the intensity of the TCR / CD3 downstream signaling reported by the luciferase expression controlled by the NFAT promoter. The schematic diagram (8A) of the assay. In the presence of HT29 or HeLa (8B) as target cells, the dose-dependent activation of the Jurkat cells transduced. Depicted is the technical mean value from three repetitions, and error bars indicate SD.

[0081] Figure 9A and 9B In the presence of CD19 +In the case of target cells, with the activation of Jurkat NFAT CD3εKO cells transduced by P329G-CAR (sorting pool) or P329G-CD3ε (sorting pool), the target cells have high target expression levels (Nalm-6) or low target expression levels (Z138) after stimulating with anti-CD19 (affinity maturation 2B11) IgG containing the P329G LALA mutation. Activation is assessed by the intensity of the TCR / CD3 downstream signaling reported by the luciferase expression controlled by the NFAT promoter. The schematic diagram (9A) of this assay. In the case of there being Z138 or Nalm-6 (9B) as target cells, the dose-dependent activation of the Jurkat cells transduced. Depicted is the technical mean value from three repetitions, and error bars indicate SD.

[0082] Figure 10A and 10B : eGFP expression in Jurkat TCRαβKO-CD4+ cells (T cell activation bioassay (TCRαβ-KO), Promega, #GA1172) after lentiviral transduction of P329G-Cαβ (10A) or P329G-CAR (10B) and pool sorting for live, eGFP-positive cells. Mock-transduced cells were used as negative controls.

[0083] Figures 11A to 11C : Surface expression of P329G-CαβTCR or P329G-CAR on Jurkat TCRαβKO-CD4+ cells (sorted pools) was examined by staining with IgG containing the P329G LALA mutation (anti-FolR1 IgG P329G LALA) and detecting binding of a secondary PE-F(ab)2 fragment anti-huIgG (F(ab)2 fragment specific) (Jackson ImmunoResearch, #109-116-097) ( Figure 11A (1)). The incorporation of VH-TCRα and VL-TCRβ chains was confirmed by staining with anti-TCRαβ-BV421 (1:50, Biolegend, #306722) and anti-CD3ε-APC (1:50, Biolegend, #300412) antibodies (11A (2, 3)). The corresponding staining results are in Figure 11B (1, 2, 3) and Figure 11C (1, 2, 3). For all stainings (1, 2, 3), staining of mock-transduced cells served as a negative control (light grey). As an additional negative control for P329G staining (1), transduced cells were also stained with secondary antibody only (staining overlapped with mock-transduced controls (light grey)).

[0084] Figure 12A and12B In the presence of FolR1 + In the case of target cells, the activation of Jurkat TCR α β KO-CD4+ cells transduced with P329G-C α β or P329G-CAR (sorting pool) has a high target expression level (HeLa) or a low target expression level (HT-29) after stimulating with anti-FolR1 (clone 16D5) IgG containing the P329G LALA mutation. Activation was assessed by the intensity of the TCR / CD3 downstream signaling reported by quantitatively expressing luciferase controlled by the IL2 promoter. The schematic diagram (12A) of the assay. In the presence of HT29 or HeLa (12B) as target cells, the dose-dependent activation of transduced Jurkat cells. Depicted is the technical mean value from three repetitions, and error bars indicate SD.

[0085] Figure 13A and 13B In the presence of CD19 + In the case of target cells, the activation of Jurkat TCR α β KO-CD4+ cells transduced with P329G-C α β or P329G-CAR (sorting pool) has a high target expression level (Nalm-6) or a low target expression level (Z138) after stimulating with anti-CD19 (affinity maturation 2B11) IgG containing the P329G LALA mutation. Activation was assessed by the intensity of the TCR / CD3 downstream signaling reported by the luciferase expression controlled by the IL2 promoter. The schematic diagram (13A) of the assay. In the presence of Z138 or Nalm-6 (13B) as target cells, the dose-dependent activation of the Jurkat cells transduced. Depicted is the technical mean value from three repetitions, and error bars indicate SD.

[0086] 14A to 14C .The pan T cells of two donors were transduced with P329G-CAR, P329G-Cαβ or P329G-CD3ε respectively. Endogenous TCRα and TCRβ chains (in the case of P329G-Cαβ constructs) and endogenous CD3ε (in the case of P329G-CD3ε constructs) were knocked out using CRISPR-Cas9. (14A) shows the expression of eGFP in two donors after transduction and knockout of respective endogenous TCR chains. The surface expression (14B) of different constructs was determined by staining with Fc-P329G LALA labeled with AF647. The staining of anti-CD3ε-PE (1:50, Biolegend, #300408) and Fc-P329G LALA-AF647 for checking the percentage of correctly assembled P329G-CD3εTCR or P329G-CαβTCR complexes is shown in (14C).

[0087] Figure 15 eGFP expression in CTLL-2 cells after lentiviral transduction with the second-generation P329G-CAR (4-1BB). Surface expression of P329G-CAR (4-1BB) was confirmed by staining with AF647-labeled Fc_P329G_LALA, showing that approximately 94% of cells expressed the receptor.

[0088] Figure 16 .Proliferation of CTLL-2 cells expressing P329G-CAR (4-1BB) incubated with Fc_P329G_LALA-IL2v / IL2v-Fc_P329G_LALA or Fc_LALA-IL2v / IL2v-Fc_LALA or Proleukin. After 72 hours of incubation, the number of cells was quantified in the CellTiter-Glo activity assay. Targeting IL2v to cells via the P329G mutation resulted in proliferation, even at low concentrations of the Fc_P329G_LALA fusion cytokine.

[0089] Figure 17 eGFP expression in primary T cells after lentiviral transduction with the second-generation P329G-CAR (CD28). Surface expression of P329G-CAR (CD28) was confirmed by staining with AF647-labeled Fc_P329G_LALA, showing that approximately 70% of cells expressed the receptor.

[0090] Figure 18 .After stimulation with Fc_P329G_LALA-IL2vQ126T or Fc_WT-IL2vQ126T, STAT5 phosphorylation (pSTAT5) of primary T cells transduced with P329G-CAR (CD28) is shown. The pSTAT5 fluorescence intensity median after gating eGFP+ (P329G-CAR+) or eGFP- (P329G-CAR-) cells is shown. Fc_P329G_LALA-IL2vQ126T is targeted to P329G-CAR T cells via P329G mutation, resulting in an EC50 difference of approximately 230 times compared to the effect of Fc_WT-IL2vQ126T on the same population. Depicted are technical means from repetitions, and error bars indicate SD. EC50 values ​​are calculated using GraphPadPrism 8.4.2 (log (agonist) vs. response--variable slope (four parameters)).

[0091] Figure 19.After stimulation with PD1-IL2v or PD1-IL2vQ126T, STAT5 phosphorylation of primary T cells transduced with P329G-CAR (CD28). The median of pSTAT5 fluorescence intensity after gating eGFP+ (P329G-CAR+) or eGFP- (P329G-CAR-) cells is shown. IL2v is further weakened by the Q126T mutation, resulting in an increase in the cis effect of the P329G mutation (about 100 times vs about 800 times). Depicted are technical means from replicates, with error bars indicating SD. EC50 values ​​are calculated using GraphPadPrism 8.4.2 (log (agonist) vs. response--variable slope (four parameters)).

[0092] Figure 20 eGFP expression in primary T cells following lentiviral transduction with different P329G-tags (based on CD8a, CD25, or IL15Ra membrane anchors). Surface expression of the P329G-tag was confirmed by staining with AF647-labeled Fc_P329G_LALA. Depending on the membrane anchor used, between 47% and 75% of cells expressed the corresponding P329G-tag construct on the cell surface.

[0093] Figure 21 STAT5 phosphorylation in primary T cells transduced with different P329G tags after stimulation with Fc_P329G_LALA-IL2v or Fc_LALA-IL2v. Median pSTAT5 fluorescence intensity is shown after gating on eGFP+ (P329G-tag+) or eGFP- (P329G-tag-) cells. The P329G-tag with IL15Ra membrane anchor exhibits the best cis-targeting window (approximately 185-fold). Depicted are technical means from replicates, with error bars indicating SD. Data were analyzed using GraphPad Prism 8.4.2 (log(agonist) vs. response - variable slope (four parameters)).

[0094] Figure 22 STAT5 phosphorylation in primary T cells transduced with different P329G tags after stimulation with PD1-IL2v or PD1-IL2vQ126T. Median pSTAT5 fluorescence intensity after gating on eGFP+ (P329G-tag+) or eGFP- (P329G-tag-) cells is shown. The P329G-tag with IL15Ra membrane anchor again showed the best cis-targeting window (approximately 75 times). Depicted are technical means from replicates, with error bars indicating SD. Data were analyzed using GraphPad Prism 8.4.2 (log(agonist) vs. response - variable slope (four parameters)).

[0095] Figure 23. CellTrace Violet proliferation assay of primary T cells expressing P329G-tag (IL15Ra) incubated with Fc_LALA-IL2v (23A), Fc_P329G_LALA-IL2v (23B) or PD1-IL2v (23C). Cells were incubated with compound for 5 days, and the proliferation of eGFP+ and eGFP- cells was assessed by flow cytometry analysis of the reduction of CellTrace Violet dye in the split population (23A, 23B and 23C). Cells were stained with AF647-labeled Fc_P329G_LALA, and eGFP expression was examined after 6 days of expansion, and the population shifted to >90% of cells expressing eGFP+ (23D) and P329G-tag (23E).

[0096] Figure 24 eGFP expression in primary T cells from two donors following CRISPR knockout of endogenous CD3ε and lentiviral transduction with P329G-CD3ε. P329G-CD3ε knockout efficiency and surface expression were examined by staining with AF647-labeled Fc_P329G_LALA and PE anti-CD3ε, showing that knockout was approximately 98% successful and that approximately 34% to 40% of cells expressed P329G-CD3ε.

[0097] Figure 25 STAT5 phosphorylation of primary T cells (donor 8) transduced with P329G-CD3ε after stimulation with Fc_P329G_LALA-IL2v or Fc_LALA-IL2v. Median pSTAT5 fluorescence intensity after gating on eGFP+ (P329G-CD3ε+) or eGFP- (P329G-CD3ε-) cells is shown. Depicted are technical means from replicates, with error bars indicating SD. EC50 values ​​were calculated using GraphPad Prism 8.4.2 (log(agonist) vs. response--variable slope (four parameters)).

[0098] Figure 26STAT5 phosphorylation of primary T cells (donor 8) transduced with P329G-CD3e after stimulation with Fc_P329G_LALA-IL2vQ126T or Fc_WT-IL2vQ126T. The median pSTAT5 fluorescence intensity after gating eGFP+ (P329G-CD3ε+) or eGFP- (P329G-CD3ε-) cells is shown. Compared with Fc_P329G_LALA-IL2v, Fc_P329G_LALA-IL2vQ126T increases the cis targeting window to approximately 90 times. Depicted are technical means from replicates, with error bars indicating SD. EC50 values ​​were calculated using GraphPad Prism 8.4.2 (log (agonist) vs. response--variable slope (four parameters)).

[0099] Figure 27. CellTrace Violet proliferation assay of primary T cells (donor 7) expressing P329G-CD3εTCR incubated with Fc_LALA-IL2v (28A) or Fc_P329G_LALA-IL2v (28B). Cells were incubated with compound for 5 days, and the proliferation of eGFP+ and eGFP- cells was assessed by flow cytometry analysis of the reduction of CellTrace Violet dye in the split population. Cells were stained with Fc_P329G_LALA labeled with AF647, and eGFP expression was examined after 5 days of amplification, and the populations shifted to >82% expression eGFP+ or approximately 75% expression P329G-CD3ε cells (28C and 28D).

[0100] Figure 28. After lentiviral transduction with different P329G receptors (P329G-CAR, P329G-tag, P329G-CD3ε, P329G-Cαβ), eGFP expression in primary T cells. The surface expression of P329G receptors was confirmed by staining with Fc_P329G_LALA labeled with AF647. According to construct, cells between 59% and 83% expressed receptors (28A) on the cell surface. KO efficiency and surface expression of P329G-CD3ε and P329G-Cαβ were assessed by staining with Fc_P329G_LALA and PE anti-CD3ε or BV421 anti-TCRαβ labeled with AF647. In the case of P329G-CD3ε, KO was about 98% successful, and about 68% of cells expressed P329G-CD3ε. P329G-Cαβ T cells showed approximately 62% correctly formed TCR complexes on the surface (28B). TMPD1 expression was assessed after reactivation of cells with human CD3 / CD28 / CD2 T cell activator (28D). PD1 expression of P329G-tagged (IL15Ra) cells is shown.

[0101] Figure 29. STAT5 phosphorylation of primary T cells transduced with P329G-CAR (CD28) after stimulation with Fc_P329G_LALA-IL2v, Fc_LALA-IL2v, IL2-Fc_P329G_LALA, IL2-Fc_LALA, Fc_P329G_LALA-IL2vQ126T, Fc_WT-IL2vQ126T, PD1-IL2v or PD1-IL2vQ126T. The median pSTAT5 fluorescence intensity after gating on eGFP+ (P329G-CAR+) or eGFP- (P329G-CAR-) cells is shown (29A). The graph was rearranged to allow direct comparison of N-terminal and C-terminal Fc-IL2v fusions, PD1-IL2v and Fc_P329G_LALA-IL2v, and Fc-fusion IL2v and IL2vQ126T (29B). Depicted are technical means from replicates, with error bars indicating SD. EC50 values ​​were calculated using GraphPad Prism 8.4.2 (log(agonist) vs. response - variable slope (four parameters)).

[0102] Figure 30. STAT5 phosphorylation in primary T cells transduced with P329G-tag (IL15Rα) after stimulation with Fc_P329G_LALA-IL2v, Fc_LALA-IL2v, IL2-Fc_P329G_LALA, IL2-Fc_LALA, Fc_P329G_LALA-IL2vQ126T, Fc_WT-IL2vQ126T, PD1-IL2v, or PD1-IL2vQ126T. Median pSTAT5 fluorescence intensity after gating on eGFP+ (P329G-tag+) or eGFP- (P329G-tag-) cells is shown (30A). The graph was rearranged to allow direct comparison of N-terminal and C-terminal Fc-IL2v fusions, PD1-IL2v and Fc_P329G_LALA-IL2v, and Fc-fusion IL2v and IL2vQ126T (30B). Depicted are technical means from replicates, with error bars indicating SD. EC50 values ​​were calculated using GraphPad Prism 8.4.2 (log(agonist) vs. response - variable slope (four parameters)).

[0103] Figure 31. STAT5 phosphorylation in primary T cells transduced with P329G-CD3ε after stimulation with Fc_P329G_LALA-IL2v, Fc_LALA-IL2v, IL2-Fc_P329G_LALA, IL2-Fc_LALA, Fc_P329G_LALA-IL2vQ126T, Fc_WT-IL2vQ126T, PD1-IL2v, or PD1-IL2vQ126T. Median pSTAT5 fluorescence intensity after gating on eGFP+ (P329G-CD3ε+) or eGFP- (P329G-CD3ε-) cells is shown (31A). Graphs were rearranged to allow direct comparison of N-terminal and C-terminal Fc-IL2v fusions, PD1-IL2v and Fc_P329G_LALA-IL2v, and Fc-fusion IL2v and IL2vQ126T (31B). Technical means from replicates are depicted, with error bars indicating SD. EC50 values ​​were calculated using GraphPadPrism 8.4.2 (log(agonist) vs. response—variable slope (four parameters)).

[0104] Figure 32. STAT5 phosphorylation in primary T cells transduced with P329G-Cαβ after stimulation with Fc_P329G_LALA-IL2v, Fc_LALA-IL2v, IL2-Fc_P329G_LALA, IL2-Fc_LALA, Fc_P329G_LALA-IL2vQ126T, Fc_WT-IL2vQ126T, PD1-IL2v, or PD1-IL2vQ126T. Median pSTAT5 fluorescence intensity after gating on eGFP+ (P329G-Cαβ+) or eGFP- (P329G-Cαβ-) cells is shown (32A). Graphs were rearranged to allow direct comparison of N-terminal and C-terminal Fc-IL2v fusions, PD1-IL2v and Fc_P329G_LALA-IL2v, and Fc-fusion IL2v and IL2vQ126T (32B). Technical means from replicates are depicted, with error bars indicating SD. EC50 values ​​were calculated using GraphPad Prism 8.4.2 (log(agonist) vs. response—variable slope (four parameters)).

[0105] Figure 33. knock out transduction with P329G-CD3ε and endogenous CD3ε, with the primary T cell of amplification with the T cell culture medium with IL2 (50IU / ml), IL7 (25ng / ml) and IL15 (50ng / ml) or 0.5nM Fc_P329G_LALA-IL2v. After 6 days, colony changes into 87% desired colony (33A) from 37% P329G-CD3ε cell. Then directly compare cell (33B) in the Incucyte killing assay using two tumor cell lines (HeLa NLR and MKN45 NLR). T cell quantity is adjusted to 10,000 eGFP+ cells per well to 10,000 target cells (E:T 1:1) for fair comparison. Observing changes in red blood cell counts over time, selectively expanded P329G-CD3ε T cells were fully functional and capable of killing target cells comparable to P329G-CD3ε T cells expanded with IL2, IL7, and IL15. This indicates that the P329G receptor is free to bind the adaptor IgG and is not blocked by Fc_P329G_LALA-IL2v. Depicted are technical means from replicates, with error bars indicating SD.

[0106] Figure 34 : Orthogonal ligands PD1-IL2v, PD1-reg-IL2v and one-arm (OA)-PD1-reg-IL2v and their interactions with GFP+ (P329G-tag+) cells vs GFP- (P329G-tag-) PD-1 低 Schematic diagram of cell binding.

[0107] Figure 35: eGFP expression and surface staining of P329G-tag (based on IL15Ra) in primary T cells after lentiviral transduction (AF647-labeled Fc_P329G_LALA). 63.9% of cells expressed GFP and P329G-tag constructs on the cell surface. PD-1 expression was stained (PD-1-PE) shortly before the pSTAT5 assay was performed. Compared to the isotype control, 4.87% of T cells were PD-1 positive (35A). STAT5 phosphorylation was examined after stimulation with PD1-IL2v, PD1-reg-IL2v, and OA-PD1-reg-IL2v. The median pSTAT5 fluorescence intensity after gating on eGFP+ (P329G-tag+) or eGFP- (P329G-tag-) cells is shown (35B).

[0108] Figure 36: Orthogonal ligands PD1-IL2v, PD1-reg-IL2v and one-arm (OA)-PD1-reg-IL2v and their interactions with GFP+ (P329G-tag+) cells vs GFP- (P329G-tag-) PD-1 高 Schematic diagram of cell binding.

[0109] Figure 37: eGFP expression and surface staining of P329G-tag (IL15Ra based) in primary T cells after lentiviral transduction (AF647 labeled Fc_P329G_LALA). 60.9% of the cells expressed GFP and P329G-tag constructs on the cell surface. For one part of the assay, the transduced T cells were reactivated using Dynabeads human T activator CD3 / CD28. For the other part of the assay, the cells were not reactivated. PD-1 expression was stained shortly before the pSTAT5 assay was performed (PD-1-PE). Compared to the isotype control, 14.7% of the T cells were PD-1 positive under non-reactivated conditions (37A), while 81.6% of the T cells were PD-1 positive under reactivated conditions (37C). STAT5 phosphorylation was examined after stimulation with PD1-IL2v, PD1-reg-IL2v and OA-PD1-reg-IL2v. Shown in PD-1 低 (37B) and PD-1 高 Median pSTAT5 fluorescence intensity after gating on eGFP+ (P329G-tag+) or eGFP- (P329G-tag-) cells under conditions (37D). DETAILED DESCRIPTION

[0110] The present inventors have produced a new orthogonal cis-targeting method that allows for the selective expansion of engineered T cells based on membrane-anchored antigen binding (MAB) polypeptides comprising an antigen binding portion that is capable of specifically binding to a CH2-CH3 region comprising an amino acid substitution P329G according to EU numbering. Exemplary embodiments include, but are not limited to, chimeric antigen receptors (CARs), TCR-based MAB polypeptides, or non-signaling tag-like MAB polypeptides. In some aspects, there is provided a recombinant Fc domain-IL2 variant (Fc-IL2v) polypeptide complex comprising a CH2-CH3 region fused to IL-2 or a variant thereof, the CH2-CH3 region comprising an amino acid substitution P329G according to EU numbering. Since naturally occurring Fc domains do not comprise the P329G mutation, the recombinant Fc-IL2v polypeptide represents an orthogonal cytokine ligand for an engineered (T) cell expressing a recombinant MAB polypeptide or a recombinant MAB polypeptide complex according to the present disclosure. The recombinant Fc-IL2v polypeptide complex according to the present invention can be used for the specific expansion of MAB polypeptide (complex) expressing T cells and the specific enrichment of such cells, resulting in reduced heterogeneity of the final cell product. The technology described herein can be translated into safe, specific and controllable CAR-T cell expansion in future patients and improve the therapeutic outcomes of cell therapies for different cancer indications (including solid tumors).

[0111] In some aspects, the present disclosure provides recombinant Fc domain-IL2 variant (Fc-IL2v) polypeptide complexes, and combinations of such Fc-IL2v polypeptide complexes with recombinant membrane-anchored antigen binding (MAB) polypeptides for use as combination therapies in treating cancer, in preventing or treating metastasis, or in stimulating an immune response or function such as T cell activity. The present disclosure further provides nucleic acids and vectors encoding such Fc-IL2v polypeptide complexes and / or recombinant MAB polypeptides, cells comprising such Fc-IL2v polypeptide complexes and / or recombinant MAB polypeptides, and compositions comprising such Fc-IL2v polypeptide complexes, recombinant MAB polypeptides, and / or cells.

[0112] More specifically, the present disclosure relates to a novel recombinant Fc-IL2v polypeptide complex comprising a variant CH2-CH3 polypeptide and an IL-2 variant polypeptide. The present disclosure further relates to a recombinant MAB polypeptide comprising an antigen-binding portion and a transmembrane domain, and the MAB polypeptide complex, wherein the antigen-binding portion specifically binds to the variant CH2-CH3 region of the Fc-IL2 polypeptide complex. Thus, cells (e.g., T cells) containing the recombinant MAB polypeptide specifically bind to the recombinant Fc-IL2v polypeptide complex and are activated.

[0113] Unexpectedly, the present disclosure demonstrates that recombinant Fc-IL2v polypeptide complexes are capable of triggering potent IL2 receptor-mediated signaling in cells (eg, T cells) comprising (eg, by expressing) a MAB polypeptide and / or MAB polypeptide complex.

[0114] Even more unexpectedly, in experiments providing a direct comparison of activation levels of T cells contacted with the novel Fc-IL2v polypeptide complexes and expressing MAB polypeptides and / or MAB polypeptide complexes, it was shown that T cells were activated to a greater extent by the novel Fc-IL2v polypeptide complexes that did not comprise any further antigen binding moieties.

[0115] Unless otherwise defined below, the terms used herein are generally as used in the art.

[0116] Methods of treatment and compositions

[0117] In some aspects, the present invention includes a method for treating a patient in need of treatment, characterized by administering to the patient a therapeutically effective amount of a combination therapy of an Fc-IL2v polypeptide complex as described herein and a recombinant MAB polypeptide and / or a recombinant MAB polypeptide complex (cells expressing them) as described herein.

[0118] Further provided are uses of an Fc-IL2v polypeptide complex as described herein and a recombinant MAB polypeptide and / or a recombinant MAB polypeptide complex (cells expressing the same) as described herein for such combination therapy.

[0119] A preferred embodiment of the present invention is a combination therapy of the Fc-IL2v polypeptide complex as described herein and the recombinant MAB polypeptide and / or the recombinant MAB polypeptide complex (cells expressing them) as described herein for the treatment of cancer or tumor.

[0120] Thus, one embodiment of the present invention is an Fc-IL2v polypeptide complex as described herein for use in combination with a recombinant MAB polypeptide and / or a recombinant MAB polypeptide complex (cells expressing the same) as described herein for treating cancer or tumors.

[0121] Another embodiment of the present invention is a recombinant MAB polypeptide and / or a recombinant MAB polypeptide complex (cells expressing them) as described herein for use in combination with an Fc-IL2v polypeptide complex as described herein for treating cancer or tumors.

[0122] In some aspects, the recombinant Fc-IL2v polypeptide complex, recombinant MAB polypeptide and / or MAB polypeptide complex, method or use as described herein further comprises administering a targeting antibody (comprising G329 in the Fc domain according to EU numbering) as further described below.

[0123] In some aspects, cancer or tumor can present antigens, such as FolR1, CEA or CD19. In other aspects, cancer or tumor can present antigens in a tumor cell environment (for example, on PD-1+T cells). As a target of combination therapy, PD-1 may be presented in a tumor cell environment (for example, in PD-1+T cells). Treatment can be of solid tumors. Treatment can be of cancer. Cancer can be selected from the group consisting of colorectal cancer, head and neck cancer, non-small cell lung cancer, breast cancer, pancreatic cancer, liver cancer and gastric cancer. Cancer can be selected from the group consisting of lung cancer, colon cancer, gastric cancer, breast cancer, head and neck cancer, skin cancer, liver cancer, kidney cancer, prostate cancer, pancreatic cancer, brain cancer and skeletal muscle cancer.

[0124] The term "cancer" as used herein can be, for example, lung cancer, non-small cell lung (NSCL) cancer, bronchoalveolar cell lung cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, gastric cancer, In some embodiments, the present invention relates to a cancer of the ovary, ovary, or ureter, including but not limited to: colon cancer, breast cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, bladder cancer, kidney cancer or ureteral cancer, renal cell carcinoma, renal pelvis cancer, mesothelioma, hepatocellular carcinoma, bile duct cancer, central nervous system (CNS) tumors, spinal axis tumors, brain stem glioma, glioblastoma multiforme, astrocytoma, neurilemoma, ependymoma, medulloblastoma, meningioma, squamous cell carcinoma, pituitary adenoma, lymphoma, lymphocytic leukemia, including refractory forms of any of the above cancers, or a combination of one or more of the above cancers. In a preferred embodiment, the cancer is breast cancer, colorectal cancer, melanoma, head and neck cancer, lung cancer, or prostate cancer. In a preferred embodiment, such cancer is breast cancer, ovarian cancer, cervical cancer, lung cancer or prostate cancer. In another preferred embodiment, such cancer is breast cancer, lung cancer, colon cancer, ovarian cancer, melanoma cancer, bladder cancer, kidney cancer, renal cancer, liver cancer, head and neck cancer, colorectal cancer, pancreatic cancer, gastric cancer, esophageal cancer, mesothelioma, prostate cancer, leukemia, lymphoma, myeloma. In a preferred embodiment, such cancer is a cancer that expresses FolR1, CEA and / or CD19.

[0125] One embodiment of the present invention is a combination of an Fc-IL2v polypeptide complex as described herein with a recombinant MAB polypeptide and / or a recombinant MAB polypeptide complex (cells expressing the same) as described herein, and optionally a targeting antibody as described herein (comprising G329 in the Fc domain according to EU numbering), for use in treating any of the above cancers or tumors. Another embodiment of the present invention is a combination of a recombinant MAB polypeptide and / or a recombinant MAB polypeptide complex (cells expressing the same) as described herein, with an Fc-IL2v polypeptide complex as described herein, and optionally a targeting antibody as described herein (comprising G329 in the Fc domain according to EU numbering), for use in treating any of the above cancers or tumors.

[0126] The present invention includes combination therapies using an Fc-IL2v polypeptide complex as described herein with a recombinant MAB polypeptide and / or a recombinant MAB polypeptide complex (cells expressing the same) and, optionally, a targeting antibody as described herein (comprising G329 in the Fc domain according to EU numbering), for the treatment of cancer.

[0127] The present invention includes combination therapy using an Fc-IL2v polypeptide complex as described herein with a recombinant MAB polypeptide and / or a recombinant MAB polypeptide complex (cells expressing the same) and, optionally, a targeting antibody as described herein (comprising G329 in the Fc domain according to EU numbering), for the prevention or treatment of metastasis.

[0128] The present invention includes combination therapies of an Fc-IL2v polypeptide complex as described herein with a recombinant MAB polypeptide and / or a recombinant MAB polypeptide complex (cells expressing the same) as described herein and, optionally, a targeting antibody as described herein (comprising G329 in the Fc domain according to EU numbering), for stimulating an immune response or function such as T cell activity.

[0129] The present invention comprises a method for treating cancer in a patient in need thereof, characterized in that an Fc-IL2v polypeptide complex as described herein is administered to the patient together with a recombinant MAB polypeptide and / or recombinant MAB complex as described herein (cells expressing the same) and optionally a targeting antibody as described herein (comprising G329 in the Fc domain according to EU numbering).

[0130] The present invention comprises a method for preventing or treating metastasis in a patient in need thereof, characterized in that an Fc-IL2v polypeptide complex as described herein is administered to the patient together with a recombinant MAB polypeptide and / or recombinant MAB complex as described herein (cells expressing the same) and optionally a targeting antibody as described herein (comprising G329 in the Fc domain according to EU numbering).

[0131] The present invention comprises a method for stimulating an immune response or function, such as T cell activity, in a patient in need thereof, characterized in that an Fc-IL2v polypeptide complex as described herein and a recombinant MAB polypeptide and / or recombinant MAB complex (cells expressing the same) as described herein and optionally a targeting antibody as described herein (comprising G329 in the Fc domain according to EU numbering) are administered to the patient.

[0132] The present invention includes an Fc-IL2v polypeptide complex as described herein for use in combination with a recombinant MAB polypeptide and / or a recombinant MAB complex (cells expressing the same) as described herein and, optionally, a targeting antibody as described herein (comprising G329 in the Fc domain according to EU numbering) for treating cancer, or alternatively for use in combination with a recombinant MAB polypeptide and / or a recombinant MAB complex (cells expressing the same) as described herein and, optionally, a targeting antibody as described herein (comprising G329 in the Fc domain according to EU numbering) for the manufacture of a medicament for treating cancer.

[0133] The present invention includes an Fc-IL2v polypeptide complex as described herein for use in combination with a recombinant MAB polypeptide and / or a recombinant MAB complex (cells expressing the same) as described herein and, optionally, a targeting antibody as described herein (comprising G329 in the Fc domain according to EU numbering) for preventing or treating metastasis, or alternatively for use in combination with a recombinant MAB polypeptide and / or a recombinant MAB complex (cells expressing the same) as described herein and, optionally, a targeting antibody as described herein (comprising G329 in the Fc domain according to EU numbering) for the manufacture of a medicament for preventing or treating metastasis.

[0134] The present invention includes an Fc-IL2v polypeptide complex as described herein for use in stimulating an immune response or function such as T cell activity in combination with a recombinant MAB polypeptide and / or a recombinant MAB complex (cells expressing the same) as described herein and, optionally, a targeting antibody as described herein (comprising G329 in the Fc domain according to EU numbering) or, alternatively, for use in the manufacture of a medicament for stimulating an immune response or function such as T cell activity in combination with a recombinant MAB polypeptide and / or a recombinant MAB complex (cells expressing the same) as described herein and, optionally, a targeting antibody as described herein (comprising G329 in the Fc domain according to EU numbering).

[0135] The present invention includes a recombinant MAB polypeptide and / or recombinant MAB complex as described herein (cells expressing the same) for use in combination with an Fc-IL2v polypeptide complex as described herein and, optionally, a targeting antibody as described herein (comprising G329 in the Fc domain according to EU numbering) for treating cancer, or alternatively for use in combination with an Fc-IL2v polypeptide complex as described herein and, optionally, a targeting antibody as described herein (comprising G329 in the Fc domain according to EU numbering) for the manufacture of a medicament for treating cancer.

[0136] Some aspects of the invention include a targeting antibody as described herein (comprising G329 in the Fc domain according to EU numbering) in combination with an Fc-IL2v polypeptide complex as described herein, and optionally a recombinant MAB polypeptide and / or a recombinant MAB complex (cells expressing the same) as described herein, for use in treating cancer, or alternatively in combination with an Fc-IL2v polypeptide complex as described herein, and optionally a recombinant MAB polypeptide and / or a recombinant MAB complex (cells expressing the same) as described herein, for use in the manufacture of a medicament for treating cancer.

[0137] In a preferred embodiment of the present invention, the recombinant Fc-IL2v polypeptide complex used in the above-mentioned combination therapy and medical use for different diseases is an Fc-IL2v polypeptide complex characterized by comprising the polypeptide sequences of SEQ ID NO: 42 and SEQ ID NO: 44 or SEQ ID NO: 41 and SEQ ID NO: 51, and the MAB polypeptide or MAB polypeptide complex used in such combination therapy is characterized by comprising the polypeptide sequences of SEQ ID NO: 63, SEQ ID NO: 67, SEQ ID NO: 71, SEQ ID NO: 146, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 154, SEQ ID NO: 222, SEQ ID NO: 235 and SEQ ID NO: 255 or SEQ ID NO: 251 and SEQ ID NO: 239.

[0138] In another aspect, the present invention provides a composition, e.g., a pharmaceutical composition, comprising an Fc-IL2v polypeptide complex as described herein and a recombinant MAB polypeptide and / or recombinant MAB complex (cells expressing the same) as described herein, and optionally a targeting antibody as described herein (comprising G329 in the Fc domain according to EU numbering), formulated together with a pharmaceutically acceptable carrier.

[0139] As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption / resorption delaying agents, etc. that are physiologically compatible. Preferably, the carrier is suitable for injection or infusion.

[0140] The compositions of the present invention can be administered by a variety of methods known in the art. As will be appreciated by those skilled in the art, the route and / or mode of administration will vary depending on the desired results.

[0141] Pharmaceutical carriers include sterile aqueous solutions or dispersions and sterile powders for preparing sterile injectable solutions or dispersions. The use of such media and agents for pharmaceutically active substances is known in the art. In addition to water, the carrier can also be, for example, an isotonic buffered saline solution.

[0142] Regardless of the route of administration selected, the compounds of the present invention, which can be used in a suitable hydrated form, and / or the pharmaceutical compositions of the present invention, can be formulated into pharmaceutical dosage forms by conventional methods known to those skilled in the art.

[0143] The actual dosage level of the active ingredient in the pharmaceutical composition of the present invention can be varied to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration without causing toxicity to the patient (effective amount). The selected dosage level will depend on a variety of pharmacokinetic factors, including the activity of the particular composition of the present invention employed, or its ester, salt, or amide, the route of administration, the time of administration, the rate of excretion of the particular compound employed, other drugs, compounds, and / or materials used in combination with the particular composition employed, the age, sex, weight, condition, general health, and previous medical history of the patient being treated, and similar factors well known in the medical field.

[0144] In one aspect, the invention provides a kit intended for use in treating a disease, the kit comprising, in the same container or separate containers, (a) an Fc-IL2v polypeptide complex as described herein, and (b) a recombinant MAB polypeptide and / or a recombinant MAB complex (cells expressing the same) as described herein, and optionally (c) a targeting antibody as described herein (comprising G329 in the Fc domain according to EU numbering), and optionally further comprising (d) a package insert comprising printed instructions directing the use of the combination therapy as a method of treating the disease. In addition, the kit may include: (a) a first container containing a composition comprising a recombinant MAB polypeptide and / or a recombinant MAB complex (cells expressing them) as described herein; (b) a second container containing a composition comprising an Fc-IL2v polypeptide complex as described herein; and optionally (c) a third container containing a composition comprising a targeting antibody as described herein (comprising G329 in the Fc domain according to EU numbering); and optionally (d) a fourth container containing a composition comprising an additional cytotoxic agent or other therapeutic agent. The kit in this embodiment of the invention may also include a package insert indicating that the composition can be used to treat a specific condition. Alternatively or in addition, the kit may further include a third (or fourth) container containing a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. It may also include other materials desired from a commercial and user perspective, including other buffers, diluents, filters, needles, and syringes.

[0145] In one aspect, the present invention provides a kit for treating a disease, the kit comprising: (a) a container comprising an Fc-IL2v polypeptide complex as described herein; and (b) a package insert comprising instructions for use of a recombinant Fc-IL2v polypeptide complex in combination therapy with a recombinant MAB polypeptide and / or a recombinant MAB complex (cells expressing the same) as described herein, and optionally a targeting antibody as described herein (comprising G329 in the Fc domain according to EU numbering) as a method for treating a disease.

[0146] In another aspect, the present invention provides a kit for treating a disease, the kit comprising: (a) a container comprising a recombinant MAB polypeptide and / or a cell expressing the recombinant MAB complex as described herein, and (b) a package insert comprising instructions for use of the recombinant MAB polypeptide (cell expressing the same) in combination therapy with a recombinant Fc-IL2v polypeptide complex as described herein and, optionally, a targeting antibody as described herein (comprising G329 in the Fc domain according to EU numbering) as a method for treating a disease.

[0147] In another aspect, the present invention provides a kit for treating a disease, the kit comprising: (a) a container comprising a targeting antibody as described herein (comprising G329 in the Fc domain according to EU numbering), and (b) a package insert comprising instructions for use of the targeting antibody (comprising G329 in the Fc domain according to EU numbering) in combination therapy with an Fc-IL2v polypeptide complex as described herein and a recombinant MAB polypeptide and / or a recombinant MAB complex (cells expressing the same) as a method for treating a disease.

[0148] In another aspect, the invention provides a medicament intended for use in a disease, the medicament comprising an Fc-IL2v polypeptide complex as described herein, wherein the medicament is for use in combination therapy with a recombinant MAB polypeptide and / or recombinant MAB complex (cells expressing the same) as described herein and optionally a targeting antibody (comprising G329 in the Fc domain according to EU numbering), and optionally comprises a package insert comprising printed instructions directing the use of the combination therapy as a method for treating the disease.

[0149] The term "treatment" or its equivalent, when applied to, for example, cancer, refers to a procedure or course of action intended to reduce or eliminate the number of cancer cells in a patient, or to alleviate the symptoms of cancer. A "treatment" of cancer or another proliferative disorder does not necessarily mean that the cancer cells or other disorder will actually be eliminated, that the number of cells or disorders will actually be reduced, or that the cancer or other disorder will actually be put into remission. Generally, a treatment for cancer may have a low probability of success but is still expected to induce an overall beneficial course of action, given the patient's medical history and estimated survival expectancy.

[0150] The terms "administered in combination" or "co-administration", "co-administration", "combination therapy" or "combination treatment" refer to the administration of a recombinant Fc-IL2v polypeptide complex as described herein and a MAB polypeptide as described herein (cells expressing them) and optionally a targeting antibody (comprising G329 in the Fc domain according to EU numbering), for example, as separate formulations / applications (or as a single formulation / application). Co-administration can be performed simultaneously or sequentially in any order, wherein preferably there is a time period during which both (or all) active agents simultaneously exert their biological activities. The active agents are co-administered simultaneously or sequentially by continuous infusion (e.g., intravenously (iv)). When two therapeutic agents are co-administered sequentially, they can be administered in two separate administrations on the same day, or one of the agents can be administered on day 1 and the second agent can be co-administered on day 2 to day 7, preferably on day 2 to day 4. Thus, in one embodiment, the term "sequentially" means within 7 days after administration of the first component, preferably 4 days after administration of the first component; and the term "concurrently" means preferably at the same time. The term "co-administered" with respect to maintenance doses of the Fc-IL2v polypeptide complex and / or MAB polypeptides (cells expressing them) and / or targeting antibody (comprising G329 in the Fc domain according to EU numbering) means that if the treatment cycle is suitable for all drugs, for example, weekly, the maintenance doses can be co-administered simultaneously. Alternatively, the maintenance doses can be co-administered sequentially, for example, doses of the Fc-IL2v polypeptide complex and MAB polypeptides (cells expressing them) and / or targeting antibody (comprising G329 in the Fc domain according to EU numbering) are given every other week.

[0151] It is understood that the recombinant Fc-IL2v polypeptide complex, MAB polypeptide (complex) (cells expressing them) and / or targeting antibody (comprising G329 in the Fc domain according to EU numbering) are administered to the patient in a "therapeutically effective amount" (or simply "effective amount"), which is the amount of the corresponding compound or combination that will elicit the biological or medical response of a tissue, system, animal or human that the researcher, veterinarian, medical doctor or other clinician is seeking.

[0152] The amount of co-administration and the timing of co-administration will depend on the type (species, sex, age, weight, etc.) and condition of the patient being treated and the severity of the disease or condition being treated. The Fc-IL2v polypeptide complex and / or MAB polypeptides (cells expressing them) and / or targeting antibody (comprising G329 in the Fc domain according to EU numbering) are suitably co-administered to the patient once or over a series of treatments, for example on the same day or the next day or at weekly intervals.

[0153] In addition to the recombinant Fc-IL2v polypeptide complexes in combination with the recombinant MAB polypeptides and / or recombinant MAB complexes (cells expressing them) and / or targeting antibodies (comprising G329 in the Fc domain according to EU numbering) as described herein, chemotherapeutic agents may also be administered.

[0154] In one embodiment, such additional chemotherapeutic agents include, but are not limited to, anti-tumor agents including alkylating agents, including: nitrogen mustards such as dichloromethane, cyclophosphamide, ifosfamide, melphalan, and chlorambucil; nitrosoureas such as carmustine (BCNU), lomustine (CCNU), and semustine (methyl CCNU); Temodal TM (temozolomide), ethyleneimine / methylmelamines such as triethylene melamine (TEM), triethylene, thiophosphoramide (thiotepa), hexamethylmelamine (HMM, hexamethylmelamine); alkyl sulfonates such as busulfan; triazines such as dacarbazine (DTIC); antimetabolites including folic acid analogs such as methotrexate and trimetrexate, pyrimidine analogs such as 5-fluorouracil (5FU), fluorodeoxyuridine, gemcitabine, cytarabine (AraC, cytarabine), 5-azacytidine, 2,2'-difluorodeoxycytidine, purine analogs such as 6-mercaptopurine, 6-thioguanine, azathioprine, T-deoxycoformycin (pentostatin), erythrohydroxynonyl adenine (EHNA), fludarabine phosphate, and 2-chlorodeoxyadenosine (cladribine, 2-CdA); natural products, including antimitotic drugs such as paclitaxel, vinca alkaloids (including vinblastine (VLB) , vincristine and vinorelbine), taxotere, estramustine and estramustine phosphate; podophyllotoxins such as etoposide and teniposide; antibiotics such as dactinomycin D, daunomycin (erythromycin), doxorubicin, mitoxantrone, idarubicin, bleomycin, plicamycin (mithramycin), mitomycin C and actinomycin; enzymes such as L-asparaginase; biological response modifiers such as interferon-α, IL-2, G-CSF and GM-CSF; Miscellaneous drugs, including platinum coordination complexes (such as oxaliplatin, cisplatin, and carboplatin), anthracenediones (such as mitoxantrone), substituted ureas (such as hydroxyurea), methylhydrazine derivatives (including N-methylhydrazine (MIH) and procarbazine), adrenocortical suppressants (such as mitotane (o,p-DDD) and aminoglutethimide); hormones and antagonists, including adrenocortical steroid antagonists, such as prednisone and its equivalents, dexamethasone, and aminoglutethimide; Gemzar TM(gemcitabine), progestogens such as hydroxyprogesterone caproate, medroxyprogesterone acetate and megestrol acetate;Estrogens such as diethylstilbestrol and ethinyl estradiol equivalents;Antiestrogens such as tamoxifen;Androgens including testosterone propionate and fluoxymesterone / equivalents;Antiandrogens such as flutamide, gonadotropin-releasing hormone analogs and leuprorelin;And nonsteroidal antiandrogens such as flutamide.Therapies targeting epigenetic mechanisms include but are not limited to histone deacetylase inhibitors, demethylating agents (e.g., Vidaza) and transcriptional inhibition release (ATRA) therapy, which can also be combined with antigen binding proteins. In one embodiment, the chemotherapeutic agent is selected from the group consisting of taxanes (e.g., paclitaxel (Taxol), docetaxel (Taxotere), modified paclitaxels (e.g., Abraxane and Opaxio), doxorubicin, sunitinib (Sutent), sorafenib (Nexavar) and other multikinase inhibitors, oxaliplatin, cisplatin and carboplatin, etoposide, gemcitabine and vinblastine. In one embodiment, the chemotherapeutic agent is selected from the group consisting of taxanes (e.g., paclitaxel (paclitaxel), docetaxel (Taxotere), modified paclitaxels (e.g., Abraxane and Opaxio). In one embodiment, the additional chemotherapeutic agent is selected from 5-fluorouracil (5-FU), leucovorin, irinotecan or oxaliplatin. In one embodiment, the chemotherapeutic agent is 5-fluorouracil, leucovorin and irinotecan (FOLFIRI). In one embodiment, the chemotherapeutic agent is 5-fluorouracil and oxaliplatin (FOLFOX).

[0155] Specific examples of combination therapies with additional chemotherapeutic agents include, for example, therapies for the treatment of breast cancer: taxanes (e.g., docetaxel or paclitaxel) or modified paclitaxels (e.g., Abraxane or Opaxio, doxorubicin), capecitabine and / or bevacizumab (Avastin); therapies for ovarian cancer using carboplatin, oxaliplatin, cisplatin, paclitaxel, doxorubicin (or modified doxorubicin (Caelyx or Doxil)), or topotecan (Hycamtin); therapies for the treatment of renal cancer using a multikinase inhibitor, MKI (Sutent, Nexavar, or 706) and / or doxorubicin; therapies for the treatment of squamous cell carcinoma using oxaliplatin, cisplatin, and / or radiation; and therapies for the treatment of lung cancer using paclitaxel and / or carboplatin.

[0156] Thus, in one embodiment, the additional chemotherapeutic agent is selected from the group consisting of taxanes (docetaxel or paclitaxel or modified paclitaxel (Abraxane or Opaxio), doxorubicin, capecitabine and / or bevacizumab for the treatment of breast cancer.

[0157] In one embodiment, the combination therapy of Fc-IL2v with recombinant MAB polypeptides and / or recombinant MAB complexes (cells expressing them) and / or targeting antibodies (comprising G329 in the Fc domain according to EU numbering) is a therapy in which no chemotherapeutic agent is administered.

[0158] The present invention also includes methods for treating a patient suffering from such a disease as described herein.

[0159] The present invention further provides a method for the manufacture of a pharmaceutical composition comprising an effective amount of an Fc-IL2v polypeptide complex according to the invention as described herein and a recombinant MAB polypeptide and / or recombinant MAB complex according to the invention (cells expressing the same) as described herein and optionally a targeting antibody according to the invention (comprising G329 in the Fc domain according to EU numbering) as described herein and a pharmaceutically acceptable carrier, and use of the recombinant Fc-IL2v polypeptide complex according to the invention and a recombinant MAB polypeptide or recombinant MAB complex (cells expressing the same) as described herein and optionally a targeting antibody according to the invention (comprising G329 in the Fc domain according to EU numbering) as described herein for such a method.

[0160] The present invention further provides the use of an effective amount of an Fc-IL2v polypeptide complex according to the invention as described herein and a recombinant MAB polypeptide and / or recombinant MAB complex according to the invention as described herein (cells expressing the same) and, optionally, a targeting antibody according to the invention as described herein (comprising G329 in the Fc domain according to EU numbering) for the manufacture of a pharmaceutical agent for treating a patient suffering from cancer, preferably together with a pharmaceutically acceptable carrier.

[0161] Each of the components of the combination therapy is explained in more detail below.

[0162] Recombinant Fc domain-IL2 variant (Fc-IL2v) polypeptide complex

[0163] Fc domain polypeptides

[0164] The recombinant Fc-IL2v polypeptide complex of the present invention comprises a variant Fc domain as further described below. The antigen-binding portion of the recombinant MAB polypeptide (complex) of the present disclosure provides binding to the variant Fc domain. The variant Fc domain according to the present disclosure comprises an amino acid sequence that comprises at least one amino acid difference relative to a reference Fc domain to which the MAB polypeptide (complex) does not bind.

[0165] As used herein, "Fc domain" refers to a polypeptide complex formed by the interaction between two polypeptides, each comprising the CH2-CH3 region of an immunoglobulin (Ig) heavy chain constant sequence.

[0166] G-type immunoglobulins (IgG) are approximately 150 kDa glycoproteins composed of two heavy chains and two light chains. From N-terminus to C-terminus, the heavy chain comprises VH, followed by a heavy chain constant region comprising three constant domains (CH1, CH2, and CH3). Similarly, the light chain comprises VL, followed by CL. Based on the heavy chain, immunoglobulins can be classified as IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgA (e.g., IgA1, IgA2), IgD, IgE, or IgM. Light chains can be either kappa (κ) or lambda (λ).

[0167] As used herein, the term "CH2 domain" refers to the amino acid sequence corresponding to the CH2 domain of an immunoglobulin (Ig). According to the EU numbering system described in Edelman et al., Proc Natl Acad Sci USA (1969) 63(1):78-85, the CH2 domain is the region of Ig formed by positions 231 to 340 of the immunoglobulin constant domain. The term "CH3 domain" refers to the amino acid sequence corresponding to the CH3 domain of an immunoglobulin (Ig). According to the EU numbering system described in Edelman et al., Proc Natl Acad Sci USA (1969) 63(1):78-85, the CH3 domain is the region of Ig formed by positions 341 to 447 of the immunoglobulin constant domain. The term "CH2-CH3 region" refers to the amino acid sequence corresponding to the CH2 and CH3 domains of an immunoglobulin (Ig). The CH2-CH3 region is the region of an Ig formed by positions 231 to 447 of an immunoglobulin constant domain according to the EU numbering system described in Edelman et al., Proc Natl Acad Sci USA (1969) 63(1):78-85.

[0168] In some embodiments, the CH2 domain, CH3 domain and / or CH2-CH3 region according to the present disclosure correspond to the CH2 domain / CH3 domain / CH2-CH3 region of IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgA (e.g., IgA1, IgA2), IgD, IgE, or IgM. In some embodiments, the CH2 domain, CH3 domain and / or CH2-CH3 region correspond to the CH2 domain / CH3 domain / CH2-CH3 region of human IgG (e.g., hIgG1, hIgG2, hIgG3, hIgG4), hIgA (e.g., hIgA1, hIgA2), hIgD, hIgE, or hIgM. In some embodiments, the CH2 domain, CH3 domain and / or CH2-CH3 region correspond to the CH2 domain / CH3 domain / CH2-CH3 region of human IgG1 allotype (e.g., G1m1, G1m2, G1m3, or G1m17).

[0169] It should be understood that the Fc domain according to the present disclosure may form part of a larger molecule comprising the Fc domain. For example, a variant Fc domain according to the present disclosure may be included in a recombinant Fc domain-IL2 variant (Fc-IL2v) polypeptide complex as further described below. In some aspects, the recombinant Fc-IL2v polypeptide complex may be further included in an antigen binding molecule (e.g., an antibody) comprising an antigen binding portion specific for a target antigen, a variant Fc domain, and an IL2 variant according to the present disclosure.

[0170] The Fc domain provides interactions with Fc receptors and other molecules of the immune system to bring functional effects. The effector functions mediated by Fc have been reviewed, for example, in Jefferis et al., Immunol Rev 1998 163: 59-76 (the entire contents of which are hereby incorporated by reference), and are brought about by: the recruitment and activation of Fc-mediated immune cells (such as macrophages, dendritic cells, neutrophils, basophils, eosinophils, platelets, mast cells, NK cells and T cells) carried out by the interaction between the Fc region and the Fc receptors expressed by the immune cells, the recruitment of complement pathway components carried out by the combination of the Fc region and complement protein C1q, and the subsequent activation of the complement cascade. Fc-mediated functions include Fc receptor binding, antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), membrane attack complex (MAC) formation, cell degranulation, cytokine and / or chemokine production, and antigen processing and presentation.

[0171] The CH2-CH3 region sequence of human IgG1 G1m1 is shown in SEQ ID NO: 1. The CH2-CH3 region sequence of human IgG1 G1m3 is shown in SEQ ID NO: 2. The CH2-CH3 region sequence of human IgG2 is shown in SEQ ID NO: 3. The CH2-CH3 region sequence of human IgG3 is shown in SEQ ID NO: 4. The CH2-CH3 region sequence of human IgG4 is shown in SEQ ID NO: 5.

[0172] Variant Fc domains according to the present disclosure comprise an amino acid sequence that comprises at least one amino acid difference relative to a reference Fc domain. For example, a "variant CH2-CH3 region" according to the present disclosure comprises an amino acid sequence that comprises at least one amino acid difference relative to a reference CH2-CH3 domain. The CH2-CH3 region sequence of human IgG1 G1m1 is shown in SEQ ID NO: 1. The CH2-CH3 region sequence of human IgG1 G1m3 is shown in SEQ ID NO: 2. The CH2-CH3 region sequence of human IgG2 is shown in SEQ ID NO: 3. The CH2-CH3 region sequence of human IgG3 is shown in SEQ ID NO: 4. The CH2-CH3 region sequence of human IgG4 is shown in SEQ ID NO: 5. In one embodiment, the reference CH2-CH3 domain comprises a sequence selected from the group consisting of SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, and SEQ ID NO 5. In some embodiments, the reference CH2-CH3 domain comprises a sequence selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 2. In a preferred embodiment, the reference CH2-CH3 domain comprises the sequence of SEQ ID NO: 1.

[0173] In some embodiments, the reference Fc domain according to the present disclosure comprises two polypeptides, wherein each polypeptide comprises a CH2-CH3 region comprising or consisting of an amino acid sequence having at least 70% sequence identity (more preferably at least one of ≥75%, ≥80%, ≥85%, ≥86%, ≥87%, ≥88%, ≥89%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% sequence identity) to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the reference Fc domain comprises two polypeptides, wherein each polypeptide comprises a CH2-CH3 region comprising SEQ ID NO: 1 or consisting thereof.

[0174] In some embodiments, the reference Fc domain comprises two polypeptides, wherein each polypeptide comprises a CH2-CH3 region comprising, or consisting of, an amino acid sequence having at least 70% sequence identity (more preferably at least one of ≥75%, ≥80%, ≥85%, ≥86%, ≥87%, ≥88%, ≥89%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% sequence identity) to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the reference Fc domain comprises two polypeptides, wherein each polypeptide comprises a CH2-CH3 region comprising, or consisting of, SEQ ID NO: 2.

[0175] In some embodiments, the reference Fc domain comprises two polypeptides, wherein each polypeptide comprises a CH2-CH3 region comprising or consisting of an amino acid sequence having at least 70% sequence identity (more preferably at least one of ≥75%, ≥80%, ≥85%, ≥86%, ≥87%, ≥88%, ≥89%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% sequence identity) to the amino acid sequence of SEQ ID NO: 3. In some embodiments, the reference Fc domain comprises two polypeptides, wherein each polypeptide comprises a CH2-CH3 region comprising SEQ ID NO: 3 or consisting thereof.

[0176] In some embodiments, the reference Fc domain comprises two polypeptides, wherein each polypeptide comprises a CH2-CH3 region comprising, or consisting of, an amino acid sequence having at least 70% sequence identity (more preferably at least one of ≥75%, ≥80%, ≥85%, ≥86%, ≥87%, ≥88%, ≥89%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% sequence identity) to the amino acid sequence of SEQ ID NO: 4. In some embodiments, the reference Fc domain comprises two polypeptides, wherein each polypeptide comprises a CH2-CH3 region comprising, or consisting of, SEQ ID NO: 4.

[0177] In some embodiments, the reference Fc domain comprises two polypeptides, wherein each polypeptide comprises a CH2-CH3 region comprising or consisting of an amino acid sequence having at least 70% sequence identity (more preferably at least one of ≥75%, ≥80%, ≥85%, ≥86%, ≥87%, ≥88%, ≥89%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% sequence identity) to the amino acid sequence of SEQ ID NO: 5. In some embodiments, the reference Fc domain comprises two polypeptides, wherein each polypeptide comprises a CH2-CH3 region comprising SEQ ID NO: 5 or consisting thereof.

[0178] A variant Fc domain according to the present disclosure may comprise amino acid differences relative to the amino acid sequence of one or both polypeptides of a reference Fc domain according to the present disclosure.

[0179] In some embodiments, the variant Fc domain according to the present disclosure comprises two polypeptides, each comprising a CH2-CH3 region, and wherein one or both of the CH2-CH3 regions comprise an amino acid sequence that is not identical to SEQ ID NO: 1. In some embodiments, the variant Fc domain according to the present disclosure comprises two polypeptides, each comprising a CH2-CH3 region, and wherein one or both of the CH2-CH3 regions comprise an amino acid sequence that has one or more (e.g., 1, 2, 3, 4, 5, or more) amino acid differences relative to SEQ ID NO: 1. In some embodiments, each CH2-CH3 region of the variant Fc domain comprises or consists of an amino acid sequence having at least 70% sequence identity (more preferably at least one of ≥75%, ≥80%, ≥85%, ≥86%, ≥87%, ≥88%, ≥89%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99% sequence identity) to the amino acid sequence of SEQ ID NO: 1, wherein one or both of the CH2-CH3 regions comprise an amino acid sequence that is not identical to SEQ ID NO: 1. In some embodiments, each CH2-CH3 region of the variant Fc domain comprises or consists of an amino acid sequence having at least 70% sequence identity (more preferably at least one of ≥75%, ≥80%, ≥85%, ≥86%, ≥87%, ≥88%, ≥89%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99% sequence identity) to the amino acid sequence of SEQ ID NO: 1, wherein one or both of the CH2-CH3 regions comprises an amino acid sequence that has one or more (e.g., 1, 2, 3, 4, 5, or more) amino acid difference(s) relative to SEQ ID NO: 1.

[0180] In some embodiments, the variant Fc domain according to the present disclosure comprises two polypeptides, each comprising a CH2-CH3 region, and wherein one or both of the CH2-CH3 regions comprise an amino acid sequence that is not identical to SEQ ID NO: 2. In some embodiments, the variant Fc domain according to the present disclosure comprises two polypeptides, each comprising a CH2-CH3 region, and wherein one or both of the CH2-CH3 regions comprise an amino acid sequence that has one or more (e.g., 1, 2, 3, 4, 5, or more) amino acid differences relative to SEQ ID NO: 2. In some embodiments, each CH2-CH3 region of the variant Fc domain comprises or consists of an amino acid sequence having at least 70% sequence identity (more preferably at least one of ≥75%, ≥80%, ≥85%, ≥86%, ≥87%, ≥88%, ≥89%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99% sequence identity) to the amino acid sequence of SEQ ID NO: 2, wherein one or both of the CH2-CH3 regions comprise an amino acid sequence that is not identical to SEQ ID NO: 2. In some embodiments, each CH2-CH3 region of the variant Fc domain comprises or consists of an amino acid sequence having at least 70% sequence identity (more preferably at least one of ≥75%, ≥80%, ≥85%, ≥86%, ≥87%, ≥88%, ≥89%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99% sequence identity) to the amino acid sequence of SEQ ID NO: 2, wherein one or both of the CH2-CH3 regions comprises an amino acid sequence that has one or more (e.g., 1, 2, 3, 4, 5, or more) amino acid difference(s) relative to SEQ ID NO: 2.

[0181] In some embodiments, the variant Fc domain according to the present disclosure comprises two polypeptides, each comprising a CH2-CH3 region, and wherein one or both of the CH2-CH3 regions comprise an amino acid sequence that is not identical to SEQ ID NO: 3. In some embodiments, the variant Fc domain according to the present disclosure comprises two polypeptides, each comprising a CH2-CH3 region, and wherein one or both of the CH2-CH3 regions comprise an amino acid sequence that has one or more (e.g., 1, 2, 3, 4, 5, or more) amino acid differences relative to SEQ ID NO: 3. In some embodiments, each CH2-CH3 region of the variant Fc domain comprises or consists of an amino acid sequence having at least 70% sequence identity (more preferably at least one of ≥75%, ≥80%, ≥85%, ≥86%, ≥87%, ≥88%, ≥89%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99% sequence identity) to the amino acid sequence of SEQ ID NO: 3, wherein one or both of the CH2-CH3 regions comprise an amino acid sequence that is not identical to SEQ ID NO: 3. In some embodiments, each CH2-CH3 region of the variant Fc domain comprises or consists of an amino acid sequence having at least 70% sequence identity (more preferably at least one of ≥75%, ≥80%, ≥85%, ≥86%, ≥87%, ≥88%, ≥89%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99% sequence identity) to the amino acid sequence of SEQ ID NO: 3, wherein one or both of the CH2-CH3 regions comprises an amino acid sequence that has one or more (e.g., 1, 2, 3, 4, 5, or more) amino acid difference(s) relative to SEQ ID NO: 3.

[0182] In some embodiments, the variant Fc domain according to the present disclosure comprises two polypeptides, each comprising a CH2-CH3 region, and wherein one or both of the CH2-CH3 regions comprise an amino acid sequence that is not identical to SEQ ID NO: 4. In some embodiments, the variant Fc domain according to the present disclosure comprises two polypeptides, each comprising a CH2-CH3 region, and wherein one or both of the CH2-CH3 regions comprise an amino acid sequence that has one or more (e.g., 1, 2, 3, 4, 5, or more) amino acid differences relative to SEQ ID NO: 4. In some embodiments, each CH2-CH3 region of the variant Fc domain comprises or consists of an amino acid sequence having at least 70% sequence identity (more preferably at least one of ≥75%, ≥80%, ≥85%, ≥86%, ≥87%, ≥88%, ≥89%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99% sequence identity) to the amino acid sequence of SEQ ID NO: 4, wherein one or both of the CH2-CH3 regions comprise an amino acid sequence that is not identical to SEQ ID NO: 4. In some embodiments, each CH2-CH3 region of the variant Fc domain comprises or consists of an amino acid sequence having at least 70% sequence identity (more preferably at least one of ≥75%, ≥80%, ≥85%, ≥86%, ≥87%, ≥88%, ≥89%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99% sequence identity) to the amino acid sequence of SEQ ID NO: 4, wherein one or both of the CH2-CH3 regions comprises an amino acid sequence that has one or more (e.g., 1, 2, 3, 4, 5, or more) amino acid difference(s) relative to SEQ ID NO: 4.

[0183] In some embodiments, the variant Fc domain according to the present disclosure comprises two polypeptides, each comprising a CH2-CH3 region, and wherein one or both of the CH2-CH3 regions comprise an amino acid sequence that is not identical to SEQ ID NO: 5. In some embodiments, the variant Fc domain according to the present disclosure comprises two polypeptides, each comprising a CH2-CH3 region, and wherein one or both of the CH2-CH3 regions comprise an amino acid sequence that has one or more (e.g., 1, 2, 3, 4, 5, or more) amino acid differences relative to SEQ ID NO: 5. In some embodiments, each CH2-CH3 region of the variant Fc domain comprises or consists of an amino acid sequence having at least 70% sequence identity (more preferably at least one of ≥75%, ≥80%, ≥85%, ≥86%, ≥87%, ≥88%, ≥89%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99% sequence identity) to the amino acid sequence of SEQ ID NO: 5, wherein one or both of the CH2-CH3 regions comprise an amino acid sequence that is not identical to SEQ ID NO: 5. In some embodiments, each CH2-CH3 region of the variant Fc domain comprises or consists of an amino acid sequence having at least 70% sequence identity (more preferably at least one of ≥75%, ≥80%, ≥85%, ≥86%, ≥87%, ≥88%, ≥89%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99% sequence identity) to the amino acid sequence of SEQ ID NO: 5, wherein one or both of the CH2-CH3 regions comprises an amino acid sequence that has one or more (e.g., 1, 2, 3, 4, 5, or more) amino acid difference(s) relative to SEQ ID NO: 5.

[0184] In some embodiments, each CH2-CH3 region of a variant Fc domain according to the present disclosure comprises amino acid differences relative to a reference Fc domain according to the present disclosure. In some embodiments, the amino acid sequences of the CH2-CH3 regions of the constituent polypeptides of the variant Fc domain according to the present disclosure are identical (i.e., they have the same amino acid sequence).

[0185] Amino acid differences in a variant Fc domain according to the present disclosure (relative to a reference Fc domain) can affect Fc-mediated functions.

[0186] Modifications of Fc domains that affect Fc-mediated function are known in the art, such as, for example, those described in Wang et al., Protein Cell (2018) 9(1): 63-73 and Saunders et al., Front Immunol. (2019) 10: 1296, both of which are incorporated herein by reference in their entireties. Exemplary Fc domain modifications known to affect Fc-mediated function are summarized in Table 1 of Wang et al., Protein Cell (2018) 9(1): 63-73 and Tables 1, 2, and 3 of Saunders et al., Front Immunol. (2019) 10: 1296. In some embodiments, a variant Fc domain of the present disclosure comprises an Fc domain comprising amino acid differences relative to a reference Fc domain (e.g., a reference Fc according to the present disclosure) that increase or decrease Fc-mediated function.

[0187] In some embodiments, the variant Fc domain comprises amino acid differences relative to a reference Fc domain that increase Fc-mediated function. In some embodiments, the variant Fc domain comprises amino acid differences relative to a reference Fc domain that increase ADCC, ADCP, and / or CDC. Thus, in some embodiments, the variant Fc domain exhibits increased levels of Fc-mediated function compared to a reference Fc domain. In some embodiments, the variant Fc domain exhibits increased ADCC, ADCP, and / or CDC compared to a reference Fc domain.

[0188] In some embodiments, the variant Fc domain comprises amino acid differences relative to a reference Fc domain, and the amino acid differences increase binding to an Fc receptor (e.g., an Fcγ receptor, e.g., FcγRI, FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa, and / or FcγRIIIb). In some embodiments, the variant Fc domain comprises amino acid differences relative to a reference Fc domain, and the amino acid differences increase binding to FcRn. In some embodiments, the variant Fc domain comprises amino acid differences relative to a reference Fc domain, and the amino acid differences increase binding to complement proteins (e.g., C1q). In some embodiments, the variant Fc domain comprises amino acid differences relative to a reference Fc domain to increase hexamerization of the antigen binding molecules comprising the variant Fc domain. In some embodiments, the Fc domain comprises amino acid differences relative to a reference Fc domain, and the amino acid differences increase the half-life of the antigen binding molecules comprising the variant Fc domain. Thus, in some embodiments, the variant Fc domain exhibits increased binding to Fc receptors (e.g., Fcγ receptors, such as FcγRI, FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa, and / or FcγRIIIb) compared to a reference Fc domain. In some embodiments, the variant Fc domain exhibits increased binding to FcRn compared to a reference Fc domain. In some embodiments, the variant Fc domain exhibits increased binding to complement proteins (e.g., C1q) compared to a reference Fc domain. In some embodiments, the antigen binding molecules comprising the variant Fc domain exhibit increased hexamerization compared to an antigen binding molecule comprising a reference Fc domain. In some embodiments, the antigen binding molecules comprising the variant Fc domain exhibit increased half-life compared to an antigen binding molecule comprising a reference Fc domain.

[0189] In some embodiments, the variant Fc domain comprises amino acid differences relative to a reference Fc domain that reduce Fc-mediated function. In some embodiments, the variant Fc domain comprises amino acid differences relative to a reference Fc domain that reduce ADCC, ADCP, and / or CDC. Thus, in some embodiments, the variant Fc domain exhibits reduced levels of Fc-mediated function compared to a reference Fc domain. In some embodiments, the variant Fc domain exhibits reduced ADCC, ADCP, and / or CDC compared to a reference Fc domain.

[0190] In some embodiments, the variant Fc domain comprises amino acid differences relative to a reference Fc domain, and the amino acid differences reduce binding to an Fc receptor (e.g., an Fcγ receptor, e.g., FcγRI, FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa, and / or FcγRIIIb). In some embodiments, the variant Fc domain comprises amino acid differences relative to a reference Fc domain, and the amino acid differences reduce binding to FcRn. In some embodiments, the variant Fc domain comprises amino acid differences relative to a reference Fc domain, and the amino acid differences reduce binding to complement proteins (e.g., C1q). In some embodiments, the variant Fc domain comprises amino acid differences relative to a reference Fc domain to increase hexamerization of the antigen binding molecules comprising the variant Fc domain. In some embodiments, the Fc domain comprises amino acid differences relative to a reference Fc domain, and the amino acid differences reduce the half-life of the antigen binding molecules comprising the variant Fc domain. Thus, in some embodiments, the variant Fc domain exhibits reduced binding to Fc receptors (e.g., Fcγ receptors, such as FcγRI, FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa, and / or FcγRIIIb) compared to a reference Fc domain. In some embodiments, the variant Fc domain exhibits reduced binding to FcRn compared to a reference Fc domain. In some embodiments, the variant Fc domain exhibits reduced binding to complement proteins (e.g., C1q) compared to a reference Fc domain. In some embodiments, the antigen binding molecules comprising the variant Fc domain exhibit reduced hexamerization compared to an antigen binding molecule comprising a reference Fc domain. In some embodiments, the antigen binding molecules comprising the variant Fc domain exhibit reduced half-life compared to an antigen binding molecule comprising a reference Fc domain.

[0191] In some embodiments, the variant Fc domain comprises a CH2-CH3 region comprising an amino acid difference at position 329 relative to the amino acid sequence of the CH2-CH3 region of a reference Fc domain. In some embodiments, the variant Fc domain comprises a CH2-CH3 region comprising amino acid differences at positions 234, 235, and 329 relative to the amino acid sequence of the CH2-CH3 region of a reference Fc domain.

[0192] In some embodiments, the variant Fc domain comprises a CH2-CH3 region comprising an amino acid difference at P329 relative to the amino acid sequence of the CH2-CH3 region of a reference Fc domain. In some embodiments, the variant Fc domain comprises a CH2-CH3 region comprising an amino acid difference at positions L234, L235, and P329 relative to the amino acid sequence of the CH2-CH3 region of a reference Fc domain.

[0193] In some embodiments, the variant Fc domain comprises a CH2-CH3 region comprising the amino acid substitution P329G relative to the amino acid sequence of the CH2-CH3 region of a reference Fc domain. In some embodiments, the variant Fc domain comprises a CH2-CH3 region comprising the amino acid substitutions L234A, L235A, and P329G relative to the amino acid sequence of the CH2-CH3 region of a reference Fc domain.

[0194] In some embodiments, the variant Fc domain according to the present disclosure comprises a polypeptide comprising a CH2-CH3 region comprising or consisting of an amino acid sequence having at least 70% sequence identity (more preferably at least one of ≥75%, ≥80%, ≥85%, ≥86%, ≥87%, ≥88%, ≥89%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% sequence identity) to the amino acid sequence of SEQ ID NO: 6 or 8, wherein the CH2-CH3 region comprises G329. In some embodiments, the variant Fc domain according to the present disclosure comprises two polypeptides, each comprising a CH2-CH3 region comprising or consisting of an amino acid sequence having at least 70% sequence identity (more preferably at least one of ≥75%, ≥80%, ≥85%, ≥86%, ≥87%, ≥88%, ≥89%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% sequence identity) to the amino acid sequence of SEQ ID NO: 6 or 8, wherein the CH2-CH3 region comprises G329. In some embodiments, the variant Fc domain according to the present disclosure comprises one or more (e.g., two) polypeptides comprising the amino acid sequence of SEQ ID NO: 6 or 8.

[0195] IL-2 pathway and IL2 variant polypeptides

[0196] In one aspect of the invention, a variant Fc domain (or variant CH2-CH3 region) as described above is fused to an IL2 variant as described below to form an Fc domain-IL2 variant (Fc-IL2v) polypeptide complex.

[0197] The ability of IL-2 to expand and activate lymphocyte and NK cell populations in vitro and in vivo explains its antitumor effects. However, as a regulatory mechanism to prevent excessive immune responses and potential autoimmunity, IL-2 causes activation-induced cell death (AICD) and renders activated T cells susceptible to Fas-mediated apoptosis.

[0198] In addition, IL-2 participates in the peripheral CD4 + CD25 + T reg Maintenance and expansion of cells (Fontenot JD, Rasmussen JP, Gavin MA et al. A function for interleukin 2in Foxp3expressing regulatory T cells. Nat Immunol. 2005; 6: 1142-1151; D'Cruz LM, Klein L. Development and function of agonist-induced CD25 + Foxp3 + regulatory T cells in the absence of interleukin 2signaling. Nat Immunol. 2005; 6:1152 1159; Maloy KJ, PowrieF. Fueling regulation: IL-2keeps CD4 + T reg cells fit.Nat Immunol.2005;6:1071-1072). These cells inhibit effector T cells from destroying themselves or their targets through cell contact or by releasing immunosuppressive cytokines such as IL-10 or transforming growth factor (TGF)-β. regDepletion of these cells has been shown to enhance IL-2-induced antitumor immunity (Imai H, Saio M, Nonaka K et al., Depletion of CD4+CD25+regulatory T cells enhances interleukin-2-induced antitumor immunity in a mouse model of colonadenocarcinoma. Cancer Sci. 2007; 98: 416-423).

[0199] During the primary and secondary expansion of CD8+T cells, IL-2 also plays an important role in the differentiation of memory CD8+T cells. IL-2 seems to be responsible for the optimal expansion and generation of effector functions after the initial antigen attack. During the contraction phase of the immune response, most antigen-specific CD8+T cells disappear due to apoptosis. The IL-2 signal can rescue CD8+T cells from cell death and provide a lasting increase in memory CD8+T cells. In the memory phase, the administration of exogenous IL-2 can increase CD8+T cell frequency. In addition, only CD8+T cells that receive IL-2 signals during the initial priming period can mediate effective secondary expansion after a new antigen attack. Therefore, the IL-2 signal during the different stages of the immune response is the key to optimizing CD8+T cell function, thereby affecting the primary and secondary responses of these T cells (Adv Exp Med Biol. 2010; 684: 28-41. The role of interleukin-2 in memory CD8 cell differentiation. Boyman O1, Cho JH, Sprent J).

[0200] Based on its antitumor efficacy, high-dose IL-2 (aldesleukin, as Therapy (marketed as IL-2) has been approved in the United States for patients with metastatic renal cell carcinoma (RCC) and malignant melanoma and in the European Union for patients with metastatic RCC. However, due to the mode of action of IL-2, systemic and non-targeted administration of IL-2 may be regThe induction of cells and AICD greatly impairs anti-tumor immunity. Another problem of systemic IL-2 treatment is relevant with the serious side effects after intravenous administration, wherein serious side effects include serious cardiovascular, pulmonary edema, liver, gastrointestinal (GI), neurological and hematological events (Proleukin (aldesleukin) Summary of Product Characteristics [SmPC]: http: / / www.medicines.org.uk / emc / medicine / 19322 / SPC / (visited on May 27, 2013)). Low-dose IL-2 regimens have been tested in patients, but the cost is suboptimal treatment results. In summary, if the shortcomings associated with its application can be overcome, then the treatment method utilizing IL-2 may be useful for cancer therapy.

[0201] In particular, mutant IL-2 (e.g., a quadruple mutant referred to as IL-2qm) is designed to overcome the limitations of wild-type IL-2 (e.g., aldesleukin) or first-generation IL-2-based immunoconjugates by eliminating binding to the IL-2Rα subunit (CD25). This mutant IL-2qm has been conjugated to a variety of tumor-targeting antibodies, such as humanized antibodies against CEA and antibodies against FAP, as described in WO 2012 / 146628 and WO 2012 / 107417. In addition, the Fc region of the antibody is modified to prevent binding to Fcγ receptors and C1q complexes. The resulting tumor-targeted IL-2 variant immunoconjugates (e.g., CEA-targeted IL-2 variant immunoconjugates and FAP-targeted IL-2 variant immunoconjugates) have been shown to eliminate tumor cells in non-clinical in vitro and in vivo experiments.

[0202] The term "IL-2" or "human IL-2" refers to human IL-2 protein, including wild-type and variants comprising one or more mutations in the amino acid sequence of wild-type IL-2, such as shown in SEQ ID NO: 38 with a C125A substitution to prevent the formation of disulfide-bridged IL-2 dimers. IL-2 can also be mutated to remove N- and / or O-glycosylation sites.

[0203] Variant or mutant IL-2 polypeptides according to the present disclosure ("IL-2 variant polypeptides" or "IL2v polypeptides") comprise an amino acid sequence that comprises at least one amino acid difference relative to a reference IL-2 polypeptide. For example, in a preferred embodiment, an IL-2 variant polypeptide according to the present disclosure comprises an amino acid sequence that comprises at least one amino acid difference relative to human IL-2 (as set forth in SEQ ID NO:40).

[0204] As described in WO 2012 / 146628, IL-2 mutants have reduced binding affinity for the α subunit of the IL-2 receptor. The α subunit (also known as CD25) forms a heterotrimeric high-affinity IL-2 receptor together with the β subunit and the γ subunit (also known as CD122 and CD132, respectively), while the dimeric receptor consisting only of the β subunit and the γ subunit is called the medium-affinity IL-2 receptor. As described in WO 2012 / 146628, IL-2 mutant polypeptides with reduced binding to the α subunit of the IL-2 receptor have a reduced ability to induce IL-2 signaling in regulatory T cells, induce less activation-induced cell death (AICD) in T cells, and have reduced in vivo toxicity characteristics compared to wild-type IL-2 polypeptides. The use of such IL-2 mutants with reduced toxicity is particularly advantageous in Fc-IL2v polypeptide complexes, which have a long serum half-life due to the presence of the Fc domain. Compared to wild-type IL-2, the IL-2 mutant may comprise at least one amino acid mutation that reduces or eliminates the affinity of the IL-2 mutant for the α subunit (CD25) of the IL-2 receptor, but retains the affinity of the IL-2 mutant for the intermediate affinity IL-2 receptor (composed of the β subunit and the γ subunit of the IL-2 receptor). One or more amino acid mutations may be amino acid substitutions. The IL-2 mutant may comprise one, two or three amino acid substitutions at one, two or three positions selected from the positions corresponding to residues 42, 45 and 72 of human IL-2 (as shown in SEQ ID NO: 40). The IL-2 mutant may comprise three amino acid substitutions at positions corresponding to residues 42, 45 and 72 of human IL-2. The IL-2 mutant may be a mutant of human IL-2. The IL-2 mutant may be a human IL-2 comprising the amino acid substitutions F42A, Y45A and L72G. The IL-2 mutant may further comprise an amino acid mutation at a position corresponding to position 3 of human IL-2, which eliminates the O-glycosylation site of IL-2. Specifically, the additional amino acid mutation is an amino acid substitution in which a threonine residue is replaced with an alanine residue. The specific IL-2 mutant that can be used in the present invention comprises four amino acid substitutions at positions corresponding to residues 3, 42, 45, and 72 of human IL-2 (as shown in SEQ ID NO: 40). The specific amino acid substitutions are T3A, F42A, Y45A, and L72G. As demonstrated in the examples of WO 2012 / 146628, the quadruple mutant IL-2 polypeptide (IL-2qm) exhibits no detectable binding to CD25, reduced ability to induce T cell apoptosis, reduced induction of T regThe invention relates to a method for the production of IL-2 in vitro and in vivo cells, and a method for the production of IL-2 in vitro and in vivo cells, and a method for the production of IL-2 in vitro and in vivo cells, and a method for the production of IL-2 in vitro and in vivo cells, and a method for the production of IL-2 in vitro and in vivo cells, and a method for the production of IL-2 in vitro and in vivo cells, and a method for the production of IL-2 in vitro and in vivo cells, and a method for the production of IL-2 in vitro and in vivo cells, and a method for the production of IL-2 in vitro and in vivo cells, and a method for the production of IL-2 in vitro and in vivo cells, and a method for the production of IL-2 in vitro and in vivo cells, and a method for the production of IL-2 in vitro and in vivo cells, and a method for the production of IL-2 in vitro and in vivo cells, and a method for the production of IL-2 in vitro and in vivo cells, and a method for the production of IL-2 in vitro and in vivo cells, and a method for the production of IL-2 in vitro and in vivo cells, and a method for the production of IL-2 in vitro and in vivo cells, and a method for the production of IL-2 in vitro and in vivo cells, and a method for the production of IL-2 in vitro and in vivo cells, and a method for the production of IL-2 in vitro and in vivo cells, and a method for the production of IL-2 in vitro and in vivo cells, and a method for the production of IL-2 in vitro and in vivo cells, reg The further IL-2 mutant may comprise the polypeptide sequence of SEQ ID NO: 39.

[0205] The recombinant Fc-IL2v polypeptide complex for use in the combination therapy described herein comprises a variant Fc domain as described above, and an IL-2 mutant, particularly a mutant of human IL-2, which has reduced binding affinity for the α subunit of the IL-2 receptor (compared to wild-type IL-2, e.g., human IL-2 as shown in SEQ ID NO: 40), such as IL-2 comprising: i) one, two or three amino acid substitutions at one, two or three positions selected from the positions corresponding to residues 42, 45 and 72 of human IL-2 as shown in SEQ ID NO: 40, e.g., three substitutions at three positions, e.g., the specific amino acid substitutions F42A, Y45A and L72G; or ii) the features as described in i) plus an amino acid substitution at a position corresponding to residue 3 of human IL-2 as shown in SEQ ID NO: 40, e.g., the specific amino acid substitution T3A; or iii) a position corresponding to residue 42, 45 and 72 of human IL-2 as shown in SEQ ID NO: 40, e.g., the specific amino acid substitution F42A, Y45A and L72G. or iv) four amino acid substitutions at positions corresponding to residues 3, 42, 45 and 72 of human IL-2 as shown in SEQ ID NO: 40, for example, the specific amino acid substitutions T3A, F42A, Y45A and L72G; or iv) five amino acid substitutions at positions corresponding to residues 3, 42, 45, 72 and 126 of human IL-2 as shown in SEQ ID NO: 40, for example, the specific amino acid substitutions T3A, F42A, Y45A, L72G and Q126T.

[0206] In some aspects of the present invention, the recombinant Fc-IL2v polypeptide complex does not include an antigen-binding portion. In the accompanying examples, it has been shown that an Fc-IL2v polypeptide complex consisting of: a polypeptide comprising a CH2-CH3 region comprising P329 according to EU numbering, fused to an IL-2 mutant; and a polypeptide comprising a CH2-CH3 region comprising P329 according to EU numbering, is able to activate T cells more potently than an Fc-IL2v polypeptide complex further comprising an antigen-binding portion. Without being bound by theory, an Fc-IL2v polypeptide complex that does not include an antigen-binding portion may have a steric advantage compared to more complex molecules. Furthermore, it may be desirable to target the recombinant Fc-IL2v polypeptide complex to cells expressing a MAB polypeptide according to the present invention solely through the interaction between the MAB antigen-binding portion and the CH2-CH3 region comprising P329 according to EU numbering.

[0207] In some aspects, the recombinant Fc-IL2v polypeptide complex does not comprise a variable fragment (Fv) portion, a single-chain Fv (scFv) portion, a fragment antigen-binding (Fab) portion, a single-chain Fab portion (scFab), a crossFab portion, a Fab' portion, a Fab'-SH portion, a F(ab')2 portion, a diabody portion, a triabody portion, a scFv-Fc portion, a minibody portion, a heavy chain only antibody (HCAb) portion, or a single domain antibody (dAb, VHH) portion.

[0208] In some aspects, the recombinant Fc-IL2v polypeptide complex does not comprise a Fab or crossFab antigen binding portion.

[0209] In some embodiments, a recombinant Fc-IL2v polypeptide complex according to the present disclosure comprises:

[0210] (i) a first polypeptide comprising a variant CH2-CH3 region comprising G329 according to EU numbering, and wherein the first polypeptide further comprises an IL-2 variant (IL2v) polypeptide comprising an IL-2 polypeptide comprising amino acid substitutions F42A, Y45A, and L72G, wherein the numbering is relative to the human IL-2 sequence SEQ ID NO: 40; and

[0211] (ii) a second polypeptide comprising a variant CH2-CH3 region comprising G329 according to EU numbering,

[0212] The recombinant Fc-IL2v polypeptide complex does not contain an antigen binding portion.

[0213] In some embodiments, a recombinant Fc-IL2v polypeptide complex according to the present disclosure comprises:

[0214] (i) a first polypeptide comprising a variant CH2-CH3 region comprising G329 according to EU numbering, and wherein the first polypeptide further comprises an IL-2 variant (IL2v) polypeptide comprising an IL-2 polypeptide comprising amino acid substitutions F42A, Y45A, and L72G, wherein the numbering is relative to the human IL-2 sequence SEQ ID NO: 40; and wherein the first polypeptide comprises a polypeptide selected from the group consisting of SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 47, and SEQ ID NO: 50. The amino acid sequences of the group consisting of ID NO: 48 have or consist of an amino acid sequence having at least 70% sequence identity (more preferably at least one of ≥75%, ≥80%, ≥85%, ≥86%, ≥87%, ≥88%, ≥89%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% sequence identity); and

[0215] (ii) a second polypeptide comprising a variant CH2-CH3 region comprising G329 according to EU numbering, wherein the second polypeptide comprises or consists of an amino acid sequence having at least 70% sequence identity (more preferably at least one of ≥75%, ≥80%, ≥85%, ≥86%, ≥87%, ≥88%, ≥89%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% sequence identity) to the amino acid sequence of SEQ ID NO: 42,

[0216] The recombinant Fc-IL2v polypeptide complex does not contain an antigen binding portion.

[0217] The first polypeptide and the second polypeptide are capable of stably associating.

[0218] In some embodiments, a recombinant Fc-IL2v polypeptide complex according to the present disclosure comprises:

[0219] (i) a first polypeptide comprising a variant CH2-CH3 region comprising G329 according to EU numbering, and wherein the first polypeptide further comprises an IL-2 variant (IL2v) polypeptide comprising an IL-2 polypeptide comprising amino acid substitutions F42A, Y45A, and L72G, wherein the numbering is relative to the human IL-2 sequence SEQ ID NO: 40; and wherein the first polypeptide comprises a polypeptide selected from the group consisting of SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, and SEQ ID NO: 53. The amino acid sequences of the group consisting of ID NO: 53 have or consist of an amino acid sequence having at least 70% sequence identity (more preferably at least one of ≥75%, ≥80%, ≥85%, ≥86%, ≥87%, ≥88%, ≥89%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% sequence identity); and

[0220] (ii) a second polypeptide comprising a variant CH2-CH3 region comprising G329 according to EU numbering, wherein the second polypeptide comprises or consists of an amino acid sequence having at least 70% sequence identity (more preferably at least one of ≥75%, ≥80%, ≥85%, ≥86%, ≥87%, ≥88%, ≥89%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% sequence identity) to the amino acid sequence of SEQ ID NO: 41,

[0221] The recombinant Fc-IL2v polypeptide complex does not contain an antigen binding portion.

[0222] The first polypeptide and the second polypeptide are capable of stably associating.

[0223] In some embodiments, a recombinant Fc-IL2v polypeptide complex according to the present disclosure comprises:

[0224] (i) a first polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:47 and SEQ ID NO:48; and

[0225] (ii) a second polypeptide comprising the amino acid sequence of SEQ ID NO: 42,

[0226] The recombinant Fc-IL2v polypeptide complex does not contain an antigen binding portion.

[0227] In some embodiments, a recombinant Fc-IL2v polypeptide complex according to the present disclosure comprises:

[0228] (i) a first polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, and SEQ ID NO: 53; and

[0229] (ii) a second polypeptide comprising the amino acid sequence of SEQ ID NO: 41,

[0230] The recombinant Fc-IL2v polypeptide complex does not contain an antigen binding portion.

[0231] In some embodiments, a recombinant Fc-IL2v polypeptide complex according to the present disclosure comprises:

[0232] (i) a first polypeptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:47 and SEQ ID NO:48; and

[0233] (ii) a second polypeptide consisting of the amino acid sequence of SEQ ID NO: 42.

[0234] In some embodiments, a recombinant Fc-IL2v polypeptide complex according to the present disclosure comprises:

[0235] (i) a first polypeptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52 and SEQ ID NO: 53; and

[0236] (ii) a second polypeptide consisting of the amino acid sequence of SEQ ID NO: 41,

[0237] Modifications that promote heterodimerization

[0238] As described herein, the Fc domain-IL2 variant (Fc-IL2v) polypeptide complex can comprise an Fc domain composed of two subunits and comprising modifications that promote heterodimerization of two different polypeptide chains as further described below. The recombinant Fc-IL2v polypeptide complex described herein can comprise an Fc domain subunit comprising a knob mutation and an Fc domain subunit comprising a hole mutation as previously described herein.

[0239] "Modifications that promote heterodimerization" are manipulations of the peptide backbone or post-translational modifications of a polypeptide that reduce or prevent the polypeptide from associating with the same polypeptide to form a homodimer. As used herein, modifications that promote heterodimerization particularly include separate modifications to each of the two polypeptides required to form a dimer, wherein the modifications complement each other to promote the association of the two polypeptides. For example, modifications that promote heterodimerization can change the structure or charge of one or both of the polypeptides required to form a dimer so as to favor their association spatially or electrostatically, respectively. Heterodimerization occurs between two different polypeptides, such as two subunits of an Fc domain, wherein the other immunoconjugate components (e.g., antigen binding moieties, effector moieties) fused to each subunit are different. In the recombinant Fc-IL2v polypeptide complex according to the present invention, the modifications that promote heterodimerization are located in the Fc domain. In some embodiments, the modifications that promote heterodimerization comprise amino acid mutations, particularly amino acid substitutions. In specific embodiments, the modifications that promote heterodimerization comprise separate amino acid mutations, particularly amino acid substitutions, to each of the two subunits of the Fc domain. The most extensive protein-protein interaction site between the two polypeptide chains of the human IgG Fc domain is in the CH3 domain of the Fc domain. Therefore, in one embodiment, the modification is located in the CH3 domain of the Fc domain. In a specific embodiment, the modification is a knob-to-hole modification, comprising a knob modification in one of the two subunits of the Fc domain and a hole modification in the other of the two subunits of the Fc domain.

[0240] Knob-and-hole structure technology is described in, for example, US 5,731,168; US 7,695,936; Ridgway et al., Prot Eng 9, 617-621 (1996) and Carter, J Immunol Meth 248, 7-15 (2001). Generally, the method involves introducing a protrusion ("knob") at the interface of a first polypeptide and introducing a corresponding cavity ("hole") in the interface of a second polypeptide so that the protrusion can be positioned in the cavity to promote the formation of heterodimers and hinder the formation of homodimers. The protrusion is constructed by replacing the small amino acid side chains from the interface of the first polypeptide with larger side chains (such as tyrosine or tryptophan). A compensatory cavity having the same or similar size as the protrusion is created in the interface of the second polypeptide by replacing the large amino acid side chains with smaller amino acid side chains (such as alanine or threonine). The protrusion and cavity can be prepared by changing the nucleic acid encoding the polypeptide, for example, by site-specific mutagenesis or by peptide synthesis. In a specific embodiment, the knob modification comprises the amino acid substitution T366W in one of the two subunits of the Fc domain, and the hole modification comprises the amino acid substitutions T366S, L368A, and Y407V in the other of the two subunits of the Fc domain. In another specific embodiment, the subunit comprising the knob-modified Fc domain further comprises the amino acid substitution S354C, and the subunit comprising the hole-modified Fc domain further comprises the amino acid substitution Y349C. The introduction of these two cysteine ​​residues results in the formation of a disulfide bridge between the two subunits of the Fc region, thereby further stabilizing the dimer (Carter, J Immunol Methods 248, 7-15 (2001)). The numbering of amino acid residues in the Fc region is according to the EU numbering system, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, MD, 1991. As used herein, a "subunit" of an Fc domain refers to one of the two polypeptides that form a dimeric Fc domain, i.e., a polypeptide comprising the C-terminal constant region of an immunoglobulin heavy chain, which polypeptide is capable of stably self-association. For example, a subunit of an IgG Fc domain comprises the IgG CH2 and IgG CH3 constant domains.

[0241] In an alternative embodiment, the modification that promotes heterodimerization of two non-identical polypeptide chains comprises a modification that mediates an electrostatic steering effect, for example as described in WO 2009 / 089004. Typically, this approach involves replacing one or more amino acid residues at the interface of the two polypeptide chains with a charged amino acid residue such that homodimer formation becomes electrostatically unfavorable but heterodimerization becomes electrostatically favorable.

[0242] IL-2 mutants with reduced binding affinity to the subunits of the IL-2 receptor can be fused to the carboxyl-terminal amino acid of the subunit of the Fc domain containing a modified knob. Without wishing to be bound by theory, fusion of the IL-2 mutant to the knob-containing subunit of the Fc domain will further minimize the production of homodimeric immunoconjugates containing two IL-2 mutant polypeptides (steric hindrance of the two knob-containing polypeptides).

[0243] Exemplary Fc domain-IL2 variant (Fc-IL2v) polypeptide complexes

[0244] In some aspects, a recombinant Fc domain-IL2 variant (Fc-IL2v) polypeptide complex is provided, comprising:

[0245] (i) a first polypeptide comprising a variant CH2-CH3 region comprising G329 according to EU numbering, and wherein the first polypeptide further comprises an IL-2 variant (IL2v) polypeptide comprising an IL-2 polypeptide comprising amino acid substitutions F42A, Y45A, and L72G, wherein the numbering is relative to the human IL-2 sequence SEQ ID NO:40, wherein the first polypeptide sequence is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a sequence selected from the group consisting of SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, and SEQ ID NO:53.

[0246] In some aspects, a recombinant Fc domain-IL2 variant (Fc-IL2v) polypeptide complex is provided, comprising

[0247] (ii) a second polypeptide comprising a variant CH2-CH3 region comprising G329 according to EU numbering, wherein the second polypeptide sequence is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:41 or SEQ ID NO:42.

[0248] In a specific aspect, an Fc-IL2v polypeptide complex is provided comprising a first polypeptide sequence comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52 and SEQ ID NO:53.

[0249] In a specific aspect, an Fc-IL2v polypeptide complex comprising a second polypeptide sequence is provided, the second polypeptide sequence comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 41 and SEQ ID NO: 42.

[0250] In some aspects, a recombinant Fc domain-IL2 variant (Fc-IL2v) polypeptide complex is provided, comprising:

[0251] (i) a first polypeptide comprising a variant CH2-CH3 region comprising G329 according to EU numbering, and wherein the first polypeptide further comprises an IL-2 variant (IL2v) polypeptide comprising an IL-2 polypeptide comprising amino acid substitutions F42A, Y45A, and L72G, wherein the numbering is relative to the human IL-2 sequence SEQ ID NO:40, wherein the first polypeptide sequence is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence selected from the group consisting of SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, and SEQ ID NO:53; and

[0252] (ii) a second polypeptide comprising a variant CH2-CH3 region comprising G329 according to EU numbering, wherein the second polypeptide sequence is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:41 or SEQ ID NO:42.

[0253] In a specific aspect, an Fc-IL2v polypeptide complex is provided, comprising:

[0254] (i) a first polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, and SEQ ID NO: 53; and

[0255] (ii) a second polypeptide comprising the amino acid sequence of SEQ ID NO: 41.

[0256] In a specific aspect, an Fc-IL2v polypeptide complex is provided, comprising:

[0257] (i) a first polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:47 and SEQ ID NO:48; and

[0258] (ii) a second polypeptide comprising the amino acid sequence of SEQ ID NO: 42.

[0259] In a preferred aspect, an Fc-IL2v polypeptide complex is provided, comprising:

[0260] (i) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 44; and

[0261] (ii) a second polypeptide comprising the amino acid sequence of SEQ ID NO: 42.

[0262] In a preferred aspect, an Fc-IL2v polypeptide complex is provided, comprising:

[0263] (i) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 51; and

[0264] (ii) a second polypeptide comprising the amino acid sequence of SEQ ID NO: 41.

[0265] The recombinant Fc-IL2v polypeptide complex does not contain an antigen binding portion.

[0266] In some embodiments, a recombinant Fc-IL2v polypeptide complex according to the present disclosure consists of:

[0267] (i) a first polypeptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:47 and SEQ ID NO:48; and

[0268] (ii) a second polypeptide consisting of the amino acid sequence of SEQ ID NO: 42.

[0269] In some embodiments, a recombinant Fc-IL2v polypeptide complex according to the present disclosure consists of:

[0270] (i) a first polypeptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52 and SEQ ID NO: 53; and

[0271] (ii) a second polypeptide consisting of the amino acid sequence of SEQ ID NO: 41,

[0272] The recombinant Fc-IL2v polypeptide complex does not contain an antigen binding portion.

[0273] In a preferred embodiment, the recombinant Fc-IL2v polypeptide complex according to the present disclosure consists of:

[0274] (i) a first polypeptide consisting of the amino acid sequence of SEQ ID NO: 43 or SEQ ID NO: 44; and

[0275] (ii) a second polypeptide consisting of the amino acid sequence of SEQ ID NO: 42.

[0276] Therefore, in a preferred embodiment of the present invention, the recombinant Fc-IL2v polypeptide complex for the above-mentioned combination therapy and medical use of different diseases is an Fc-IL2v polypeptide complex, characterized by consisting of the polypeptide sequences of SEQ ID NO: 42 and SEQ ID NO: 44 or SEQ ID NO: 42 and SEQ ID NO: 43.

[0277] PD-1 targeting Fc-IL2v peptide complex

[0278] In some aspects, the recombinant Fc-IL2v polypeptide complex comprises at least one antigen binding portion that binds to PD-1 ("PD1-targeted Fc-IL2v polypeptide complex"). The PD-1-targeted Fc-IL2v polypeptide complex can be prepared as described in the examples of WO 2018 / 184964. Programmed death 1 receptor (PD-1) (CD279) and its ligand binding partners PD-L1 (B7-H1, CD274) and PD-L2 (B7-DC, CD273) provide important negative co-stimulatory signals that regulate T cell activation. PD-1 knockout (Pdcd1- / -) reveals the negative regulatory effects of PD-1, which is prone to autoimmunity. Nishimura et al., Immunity 11:141-51 (1999); Nishimura et al., Science 291:319-22 (2001). PD-1 is related to CD28 and CTLA-4, but lacks the membrane-proximal cysteine ​​that allows homodimerization. The cytoplasmic domain of PD-1 contains an immunoreceptor tyrosine-based inhibitory motif (ITIM, V / IxYxxL / V). PD-1 only binds to PD-L1 and PD-L2. Freeman et al., J. Exp. Med. 192: 1-9 (2000); Dong et al., Nature Med. 5: 1365-1369 (1999); Latchman et al., Nature Immunol. 2: 261-268 (2001); Tseng et al., J. Exp. Med. 193: 839-846 (2001).

[0279] PD-1 can be expressed on T cells, B cells, natural killer T cells, activated monocytes and dendritic cells (DC). + and CD8 +T cells, B cells and bone marrow cells express, but not by unstimulated cells. This is in contrast to the more restricted expression of CD28 and CTLA-4 (Nishimura et al., Int. Immunol. 8: 773-80 (1996); Boettler et al., J. Virol. 80: 3532-40 (2006)). At least four PD-1 variants were cloned from activated human T cells, including transcripts lacking (i) exon 2, (ii) exon 3, (iii) exons 2 and 3 or (iv) exons 2 to 4 (Nielsen et al., Cell. Immunol. 235: 109-16 (2005)). Except for PD-1Δex3, the expression levels of all variants in resting peripheral blood mononuclear cells (PBMCs) were similar to those of full-length PD-1. When human T cells were activated with anti-CD3 and anti-CD28, the expression of all variants was significantly induced. The PD-1Δex3 variant lacks a transmembrane domain and, similar to soluble CTLA-4, plays an important role in autoimmunity (Ueda et al., Nature 423:506-11 (2003)). This variant is enriched in the synovial fluid and serum of patients with rheumatoid arthritis. Wan et al., J. Immunol. 177:8844-50 (2006).

[0280] The expression patterns of the two PD-1 ligands are different. PD-L1 is constitutively expressed on mouse T cells and B cells, CD, macrophages, mesenchymal stem cells and bone marrow-derived mast cells (Yamazaki et al., J. Immunol. 169: 5538-45 (2002)). PD-L1 is expressed on a variety of non-hematopoietic cells (e.g., cornea, lung, vascular epithelium, liver non-parenchymal cells, mesenchymal stem cells, pancreatic islets, placental syncytiotrophoblasts, keratinocytes, etc.) (Keir et al., Annu. Rev. Immunol. 26: 677-704 (2008)) and is upregulated on many cell types after activation. Both type I and type II interferons (IFN's) upregulate PD-L1 (Eppihimer et al., Microcirculation 9:133-45 (2002); Schreiner et al., J. Neuroimmunol. 155:172-82 (2004)). When MyD88, TRAF6, and MEK are inhibited, PD-L1 expression in cell lines is reduced (Liu et al., Blood 110:296-304 (2007)). JAK2 is also involved in the induction of PD-L1 (Lee et al., FEBSL et al., 580:755-62 (2006); Liu et al., Blood 110:296-304 (2007)). Loss or inhibition of phosphatase and tensin homolog (PTEN), a cellular phosphatase that modifies phosphatidylinositol 3-kinase (PI3K) and Akt signaling, increases posttranscriptional PD-L1 expression in cancer (Parsa et al., Nat. Med. 13:84-88 (2007)).

[0281] The expression of PD-L2 is more restricted than that of PD-L1. PD-L2 is induced to express on DC, macrophages and bone marrow-derived mast cells. PD-L2 is also expressed on about half to two-thirds of resting peritoneal B1 cells, but not on conventional B2 B cells (Zhong et al., Eur. J. Immunol. 37: 2405-10 (2007)). PD-L2+B1 cells bind phosphatidylcholine and may be important for innate immune responses to bacterial antigens. The induction of PD-L2 by IFN-γ is partially dependent on NF-κB (Liang et al., Eur. J. Immunol. 33: 2706-16 (2003)). PD-L2 can also be induced on monocytes and macrophages by GM-CF, IL-4, and IFN-γ (Yamazaki et al., J. Immunol. 169:5538-45 (2002); Loke et al., PNAS 100:5336-41 (2003)).

[0282] PD-1 signaling generally has a greater effect on the production of cytokines than on cell proliferation, and has a significant effect on the production of IFN-γ, TNF-α, and IL-2. PD-1-mediated inhibitory signaling also depends on the intensity of TCR signaling, and greater inhibition is delivered under low-level TCR stimulation. This reduction can be overcome by the co-stimulation of CD28 (Freeman et al., J. Exp. Med. 192: 1027-34 (2000)) or the presence of IL-2 (Carter et al., Eur. J. Immunol. 32: 634-43 (2002)).

[0283] There is increasing evidence that signal transduction by PD-L1 and PD-L2 may be bidirectional. That is, in addition to modifying TCR or BCR signal transduction, signal transduction can also be delivered back to cells expressing PD-L1 and PD-L2. Although treatment of dendritic cells with natural human anti-PD-L2 antibodies isolated from patients with Waldenstrom's macroglobulinemia did not find upregulation of MHC II or B7 costimulatory molecules, such cells did produce more proinflammatory cytokines, particularly TNF-α and IL-6, and stimulate T cell proliferation (Nguyen et al., J. Exp. Med. 196: 1393-98 (2002)). Treatment of mice with this antibody also (1) enhanced resistance to transplanted b16 melanoma and rapidly induced tumor-specific CTLs (Radhakrishnan et al., J. Immunol. 170:1830-38 (2003); Radhakrishnan et al., Cancer Res. 64:4965-72 (2004); Heckman et al., Eur. J. Immunol. 37:1827-35 (2007)); and (2) blocked the development of airway inflammatory disease in a mouse model of allergic asthma (Radhakrishnan et al., J. Immunol. 173:1360-65 (2004); Radhakrishnan et al., J. Allergy Clin. Immunol. 116:668-74 (2005)).

[0284] Further evidence for reverse signaling in dendritic cells ("DCs") comes from studies of bone marrow-derived DCs cultured with soluble PD-1 (the PD-1 EC domain fused to the Ig constant region—"s-PD-1") (Kuipers et al., Eur. J. Immunol. 36:2472-82 (2006)). This sPD-1 inhibits DC activation and increases IL-10 production in a reversible manner by administration of anti-PD-1.

[0285] In addition, multiple studies have shown that PD-L1 or PD-L2 receptors are independent of PD-1. B7.1 has been identified as a binding partner of PD-L1 (Butte et al., Immunity 27:111-22 (2007)). Chemical cross-linking studies have shown that PD-L1 and B7.1 can interact through their IgV-like domains. The B7.1:PD-L1 interaction can induce inhibitory signals on T cells. B7.1 connects PD-L1 to CD4 + On T cells, PD-L1 connects B7.1 to CD4 + On T cells, inhibitory signals are transmitted. When stimulated with anti-CD3 plus B7.1-coated microbeads, T cells lacking CD28 and CTLA-4 exhibited reduced proliferation and cytokine production. In T cells lacking all B7.1 receptors (i.e., CD28, CTLA-4, and PD-L1), anti-CD3 plus B7.1-coated microbeads no longer inhibited T cell proliferation and cytokine production. This suggests that in the absence of CD28 and CTLA-4, B7.1 exerts a specific effect on T cells through PD-L1. Similarly, T cells lacking PD-1 exhibited reduced proliferation and cytokine production upon stimulation with anti-CD3 plus PD-L1-coated microbeads, demonstrating the inhibitory effect of PD-L1 on T cells through B7.1. When T cells lack all known PD-L1 receptors (i.e., lack PD-1 and B7.1), T cell proliferation was no longer impaired by anti-CD3 plus PD-L1-coated microbeads. Therefore, PD-L1 can exert its inhibitory effect on T cells through either B7.1 or PD-1.

[0286] The direct interaction between B7.1 and PD-L1 demonstrates that the current understanding of costimulation is incomplete and emphasizes the importance of the expression of these molecules on T cells. - / - Studies on T cells have shown that PD-L1 on T cells can downregulate the production of T cell cytokines (Latchman et al., Proc. Natl. Acad. Sci. USA 101: 10691-96 (2004)). Since both PD-L1 and B7.1 are expressed on T cells, B cells, DCs, and macrophages, there may be directional interactions between B7.1 and PD-L1 on these cell types. In addition, PD-L1 on non-hematopoietic cells may interact with B7.1 and PD-1 on T cells, which raises the question of whether PD-L1 is involved in its regulation. One possible explanation for the inhibitory effect of the B7.1: PD-L1 interaction is that T cell PD-L1 may capture or isolate the interaction between APC B7.1 and CD28.

[0287] As a result, antagonism of signaling through PD-L1, including blocking the interaction of PD-L1 with PD-1, B7.1, or both, thereby preventing PD-L1 from sending negative co-stimulatory signals to T cells and other antigen-presenting cells, may enhance immune responses to infection (e.g., acute and chronic) and tumor immunity. In addition, the anti-PD-L1 antibodies of the present invention can be combined with antagonists of other components of PD-1:PD-L1 signaling, such as antagonist anti-PD-1 and anti-PD-L2 antibodies.

[0288] The ability of IL-2 to expand and activate lymphocytes and natural killer (NK) cells is the basis of IL-2's anti-tumor activity. IL-2 mutants designed to abrogate IL-2 binding to the IL-2α subunit (CD25) overcome this limitation of IL-2 and have been shown to eliminate tumor cells as part of tumor-targeted IL-2 variant immunoconjugates, such as CEA-targeted IL-2 variant immunoconjugates or FAP-targeted IL-2 variant immunoconjugates.

[0289] The recombinant Fc-IL2v polypeptide complex used in the combination therapy described herein may comprise an antibody, or an antigen-binding fragment thereof, that binds to PD-1 on an immune cell expressing PD-1 (particularly a T cell) or in the environment of a tumor cell, and an IL-2 mutant, particularly a mutant of human IL-2, which has reduced binding affinity for the α subunit of the IL-2 receptor (compared to wild-type IL-2, e.g., human IL-2 as set forth in SEQ ID NO: 40), such as IL-2 comprising: i) one, two or three amino acid substitutions at one, two or three positions selected from the group consisting of residues 42, 45 and 72 of human IL-2 as set forth in SEQ ID NO: 40, e.g., three substitutions at three positions, e.g., the specific amino acid substitutions F42A, Y45A and L72G; or ii) the features as described in i) plus an amino acid substitution at a position corresponding to residue 3 of human IL-2 as set forth in SEQ ID NO: 40, e.g., the specific amino acid substitution T3A; or iii) a position corresponding to residue 42, 45 and 72 of human IL-2 as set forth in SEQ ID NO: 40. Four amino acid substitutions at residues 3, 42, 45, and 72 of human IL-2 shown in NO: 40, for example, specific amino acid substitutions T3A, F42A, Y45A, and L72G.

[0290] The recombinant Fc-IL2v polypeptide complex used in the combination therapy described herein may comprise the heavy chain variable domain and the light chain variable domain of an antibody that binds to PD-1 presented on an immune cell, particularly a T cell, or in the context of a tumor cell, and an Fc domain composed of two subunits and comprising modifications that promote heterodimerization of two non-identical polypeptide chains, and an IL-2 mutant, particularly a mutant of human IL-2, which has reduced binding affinity for the α subunit of the IL-2 receptor (compared to wild-type IL-2, e.g., human IL-2 as set forth in SEQ ID NO: 40), such as IL-2 comprising: i) one, two or three amino acid substitutions at one, two or three positions selected from the group consisting of residues 42, 45 and 72 of human IL-2 as set forth in SEQ ID NO: 40, e.g., three substitutions at three positions, e.g., the specific amino acid substitutions F42A, Y45A and L72G; or ii) the features as described in i) plus the addition of the following amino acid substitutions at positions corresponding to residues 42, 45 and 72 of human IL-2 as set forth in SEQ ID NO: 40: an amino acid substitution at residue 3 of human IL-2 as shown in SEQ ID NO:40, for example, the specific amino acid substitution T3A; or iii) four amino acid substitutions at positions corresponding to residues 3, 42, 45 and 72 of human IL-2 as shown in SEQ ID NO:40, for example, the specific amino acid substitutions T3A, F42A, Y45A and L72G.

[0291] The Fc-IL2v polypeptide complex for use in combination therapy may comprise: a) the heavy chain variable domain VH of SEQ ID NO: 36 and the light chain variable domain VL of SEQ ID NO: 37, and the polypeptide sequence of SEQ ID NO: 38; or the heavy chain variable domain VH of SEQ ID NO: 36 and the light chain variable domain VL of SEQ ID NO: 37, and the polypeptide sequence of SEQ ID NO: 39; or c) the polypeptide sequence of SEQ ID NO: 54 or SEQ ID NO: 55 or SEQ ID NO: 56, or d) the polypeptide sequence of SEQ ID NO: 58, SEQ ID NO: 59 and SEQ ID NO: 60.

[0292] In some embodiments, the recombinant Fc-IL2v polypeptide complex for use in combination therapy comprises the polypeptide sequences of SEQ ID NO:54, SEQ ID NO:55, and SEQ ID NO:56.

[0293] These PD1 targeting Fc-IL2v polypeptide complexes, together with their components of the antigen binding portion, Fc domain, and effector portion, are described as examples of immunoconjugates described in WO 2018 / 184964. For example, a specific immunoconjugate based on the anti-CEA antibody CH1A1A 98 / 99 2F1 and the IL-2 quadruple mutant (qm), "PD-1 targeting IgG-IL-2qm fusion protein," is described, for example, in Examples 1 and 2 of WO 2018 / 184964.

[0294] In a preferred embodiment, PD-1 targeting of the recombinant Fc-IL2v polypeptide complex can be achieved by targeting PD-1, as described in WO 2018 / 1184964. PD-1 targeting can be achieved using an anti-PD-1 antibody or antigen-binding fragment thereof. The anti-PD-1 antibody may comprise a heavy chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO: 36, or a variant thereof that retains functionality. The anti-PD-1 antibody may comprise a light chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO: 37, or a variant thereof that retains functionality. The anti-PD-1 antibody may comprise a heavy chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 36 or a variant thereof that retains functionality, and a light chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 37 or a variant thereof that retains functionality. The anti-PD-1 antibody may comprise a heavy chain variable region sequence of SEQ ID NO: 36 and a light chain variable region sequence of SEQ ID NO: 37.

[0295] The recombinant Fc-IL2v polypeptide complex may comprise a polypeptide sequence selected from the group consisting of SEQ ID NO: 54, SEQ ID NO: 55, and SEQ ID NO: 56, or variants thereof that retain functionality (e.g., IL2v comprises an additional amino acid substitution at position corresponding to 126 of human IL-2 (set forth in SEQ ID NO: 40), wherein the amino acid substitution is Q126T). The recombinant Fc-IL2v polypeptide complex may comprise a polypeptide sequence in which a Fab heavy chain specific for PD-1 shares a carboxy-terminal peptide bond with an Fc domain subunit comprising a hole modification. The recombinant Fc-IL2v polypeptide complex may comprise the polypeptide sequence of SEQ ID NO: 54 or SEQ ID NO: 55, or variants thereof that retain functionality. The recombinant Fc-IL2v polypeptide complex may comprise a Fab light chain specific for PD-1. The recombinant Fc-IL2v polypeptide complex may comprise the polypeptide sequence of SEQ ID NO: 56, or variants thereof that retain functionality. The polypeptides may be covalently linked, for example, via a disulfide bond. The Fc domain polypeptide chain may comprise the amino acid substitutions L234A, L235A, and P329G (which may be referred to as LALA P329G).

[0296] As described in WO 2018 / 184964, the recombinant Fc-IL2v polypeptide complex can be a PD-1 targeting IgG-IL-2qm fusion protein having the sequence shown in SEQ ID NOs: 54, 55, and 56 (as described, for example, in Example 1 of WO 2018 / 184964). The recombinant Fc-IL2v polypeptide complex having the sequence shown in SEQ ID NOs: 54, 55, and 56 is referred to herein as "PD1-IL2v". The recombinant Fc-IL2v polypeptide complex having the sequence shown in SEQ ID NOs: 58, 59, and 60 is referred to herein as "muPD1-IL2v", which is a murine surrogate.

[0297] The recombinant Fc-IL2v polypeptide complex used in the combination therapy described herein can include antibodies that bind to antigens presented on immune cells (particularly T cells) or in the tumor cell environment, and IL-2 mutants with reduced binding affinity to subunits of the IL-2 receptor. The recombinant Fc-IL2v polypeptide complex can be essentially composed of antibodies that bind to PD-1 presented on immune cells (particularly T cells) or in the tumor cell environment, and IL-2 mutants with reduced binding affinity to subunits of the IL-2 receptor. The antibody can be an IgG antibody, particularly an IgG1 antibody. The recombinant Fc-IL2v polypeptide complex can include a single IL-2 mutant (i.e., there is no more than one IL-2 mutant portion) with reduced binding affinity to subunits of the IL-2 receptor.

[0298] In one embodiment, the recombinant Fc-IL2v polypeptide complex comprises or consists of a polypeptide comprising an amino acid sequence at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 322; a polypeptide comprising an amino acid sequence at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 323; a polypeptide comprising an amino acid sequence at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 324; and a polypeptide comprising an amino acid sequence at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 325.

[0299] In a preferred embodiment, the recombinant Fc-IL2v polypeptide complex consists of a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 322, a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 323, a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 324, and a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 325.

[0300] In one embodiment, the recombinant Fc-IL2v polypeptide complex comprises or consists of a polypeptide comprising an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 326; a polypeptide comprising an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 327; and a polypeptide comprising an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 328.

[0301] In a preferred embodiment, the recombinant Fc-IL2v polypeptide complex consists of a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 326, a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 327, and a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 328.

[0302] Membrane-anchored antigen-binding (MAB) peptides and MAB peptide complexes

[0303] As used herein, a "membrane-anchored antigen binding polypeptide" or "MAB polypeptide" refers to a polypeptide comprising an antigen binding moiety in combination with a variant CH2-CH3 region according to the present disclosure comprising G329 according to EU numbering and a transmembrane domain.

[0304] A "MAB polypeptide complex" according to the present disclosure comprises at least two MAB polypeptides according to the present disclosure, wherein the two MAB polypeptides form an antigen binding portion bound to a variant CH2-CH3 region according to the present disclosure comprising G329 according to EU numbering. At least one of the at least two MAB polypeptides comprises a transmembrane domain.

[0305] Exemplary configurations of MAB polypeptides (complexes) are depicted in Figures 1 and 4.

[0306] For example, Figure 1A Depicted are two MAB polypeptides according to the present disclosure composed of an antigen binding portion that binds to a variant CH2-CH3 region fused to a CD3-TCR complex polypeptide CD3ε comprising G329 that binds according to EU numbering, wherein the two MAB polypeptides are integrated into a TCR complex.

[0307] For example, Figure 1B Depicted are two MAB polypeptides forming a MAB polypeptide complex according to the present disclosure, consisting of an antigen binding portion comprising a variant CH2-CH3 region fused to the CD3-TCR complex polypeptides TCRα and TCRβ bound to G329 according to EU numbering.

[0308] For example, Figure 1C MAB polypeptides according to the present disclosure are described, which consist of an antigen binding portion fused to a variant CH2-CH3 region comprising G329 bound according to EU numbering and to the transmembrane and intracellular signaling domains of a chimeric antigen receptor (CAR).

[0309] For example, Figure 1D MAB polypeptides according to the present disclosure are described which consist of an antigen binding portion comprising a variant CH2-CH3 region fused to a transmembrane domain that binds to G329 according to EU numbering.

[0310] The MAB polypeptides (complexes) of the present disclosure comprise an antigen binding portion or component thereof as further described below. The primary function of the antigen binding portion is to provide binding to the variant CH2-CH3 domain of the recombinant Fc-IL2v polypeptide complex, as described above.

[0311] The MAB polypeptides (complexes) of the present disclosure further comprise a transmembrane domain as further described below. The primary function of a transmembrane domain is to anchor the MAB polypeptide (complex) to the plasma membrane of a MAB-expressing cell (e.g., a T cell). Thus, the transmembrane domain restricts the activity of the recombinant Fc-IL2v polypeptide complex described above to the target cell (e.g., a T cell expressing the recombinant MAB polypeptide and / or the recombinant MAB complex). In the context of the present invention, any transmembrane domain of a transmembrane protein specifically defined by the CD-nomenclature can be used to generate the antigen-binding receptors of the present invention. Additional specific transmembrane domains are described below.

[0312] In some aspects, the MAB polypeptide further comprises an intracellular signaling domain, as further described below.

[0313] MAB polypeptides include, but are not limited to, chimeric antigen receptors (CARs), recombinant T cell receptors (TCRs), and non-signaling tags, as shown in FIG1 .

[0314] P329G antigen binding portion

[0315] "Antigen binding portion" includes antibodies (i.e., immunoglobulins (Ig)) and antigen binding fragments and derivatives thereof. In some embodiments, the antigen binding portion according to the present disclosure comprises or consists of a monoclonal antibody, a monospecific antibody, a multispecific (e.g., bispecific, trispecific, etc.) antibody, a variable fragment (Fv) portion, a single-chain Fv (scFv) portion, a fragment antigen binding (Fab) portion, a single-chain Fab portion (scFab), a crossFab portion, a Fab' portion, a Fab'-SH portion, a F(ab')2 portion, a diabody portion, a triabody portion, a scFv-Fc portion, a minibody portion, a heavy chain antibody (HCAb) portion, or a single domain antibody (dAb, VHH) portion.

[0316] Antigen binding moieties according to the present disclosure also include additional target antigen binding peptides / polypeptides, such as peptide aptamers, thioredoxins, anticalins, Kunitz domains, avimers, knottins, fynomers, atrimers, DARPins, affibodies, affilins, armadillo repeat proteins (ArmRPs), OBodys, and adnectins (reviewed, for example, in Reverdatto et al., Curr Top Med Chem. 2015; 15(12): 1082–1101, the entire contents of which are incorporated herein by reference (see also, for example, Boersma et al., J Biol Chem (2011) 286: 41273-85 and Emanuel et al., Mabs (2011) 3: 38-48)). Antigen binding moieties according to the present disclosure also include target antigen binding nucleic acids, such as nucleic acid aptamers (e.g., reviewed in Zhou and Rossi Nat Rev Drug Discov. 2017 16(3):181-202). Antigen binding moieties according to the present disclosure also include target antigen binding small molecules (e.g., low molecular weight (<1000 Daltons, typically between about 300 and 700 Daltons) organic compounds).

[0317] The antigen binding portion of the MAB polypeptides of the present disclosure is capable of binding to the variant Fc domains according to the present disclosure. The antigen binding portion capable of binding to the variant Fc domains according to the present disclosure may also be described as an antigen binding portion that binds to the variant Fc domains according to the present disclosure.

[0318] The antigen binding portions described herein preferably exhibit specific binding to the variant Fc domains according to the present disclosure. As used herein, "specific binding" refers to binding that is selective for a target antigen and can be distinguished from non-specific binding to non-target antigens. An antigen binding portion that specifically binds to a given target antigen preferably binds to the target antigen with greater affinity and / or for a longer duration than when it binds to other non-target antigens.

[0319] The ability of a given moiety to specifically bind to a given target antigen can be determined by analysis according to methods known in the art, such as by ELISA, surface plasmon resonance (SPR; see, e.g., Hearty et al., Methods Mol Biol (2012) 907: 411-442), biolayer interferometry (BLI; see, e.g., Lad et al., (2015) J Biomol Screen 20(4): 498-507), flow cytometry, or by radiolabeled antigen binding assay (RIA) enzyme-linked immunosorbent assay. By such analysis, binding to a given target antigen can be measured and quantified. In some embodiments, the level of binding can be the response detected in a given assay.

[0320] In some embodiments, the antigen binding portions described herein bind with affinities (i.e., K in the micromolar range). D =9.9x 10 -4 to 1x 10 -6 M) (e.g., as determined by SPR or BLI) binds to a variant Fc domain according to the present disclosure. In some embodiments, the antigen binding portions described herein bind with submicromolar affinity (i.e., K D <1x 10 -6 M) is combined with a variant Fc domain according to the present disclosure. In some embodiments, the antigen binding portions described herein bind with an affinity in the nanomolar range (i.e., K D =9.9x 10 -7 to 1x 10 -9 M) binds to a variant Fc domain according to the present disclosure. In some embodiments, the antigen binding portions described herein bind with subnanomolar affinity (i.e., K D <1x10 -9 M) binds to a variant Fc domain according to the present disclosure. In some embodiments, the antigen binding portions described herein bind with an affinity in the picomolar range (i.e., K D =9.9x10 -10 to 1x 10 -12 M) binds to a variant Fc domain according to the present disclosure. In some embodiments, the antigen binding portions described herein bind with sub-picomolar affinities (i.e., K D <1x 10 -12 M) in combination with a variant Fc domain according to the disclosure.

[0321] The antigen binding portion of the recombinant MAB polypeptides according to the present disclosure preferably does not exhibit specific binding to a reference Fc domain according to the present disclosure. In some embodiments, the antigen binding portion does not bind to, or exhibits substantially no binding to, a reference Fc domain according to the present disclosure.

[0322] An antigen-binding portion that "does not bind" or "exhibits substantially no binding" to a given antigen exhibits a level of binding to the given antigen that is similar to the level of binding to an antigen to which the antigen-binding portion is known not to bind or is known to non-specifically bind (e.g., a non-target antigen). In some embodiments, the level of binding to a given antigen by an antigen-binding portion that does not bind or exhibits substantially no binding is ≥0.5-fold and ≤2-fold (e.g., one of ≥0.75-fold and ≤1.5-fold, ≥0.8-fold and ≤1.4-fold, ≥0.85-fold and ≤1.3-fold, ≥0.9-fold and ≤1.2-fold, ≥0.95-fold and ≤1.1-fold) relative to the level of binding exhibited by the antigen-binding portion to an antigen to which the antigen-binding portion is known not to bind or is known to non-specifically bind (e.g., a non-target antigen).

[0323] In some embodiments, the level of binding of the antigen binding moiety to a reference Fc domain according to the present disclosure is ≤ 10% of the binding of the antigen binding moiety to a variant Fc domain according to the present disclosure, as determined, for example, by ELISA, SPR, BLI, or RIA. In some embodiments, the antigen binding moiety binds to a reference Fc domain according to the present disclosure at a level that is ≤ 10% of the binding of the antigen binding moiety to a variant Fc domain according to the present disclosure, as determined, for example, by ELISA, SPR, BLI, or RIA. D ; for example, as determined by SPR or BLI) at least 0.1 orders of magnitude greater K D in combination with a reference Fc domain according to the present disclosure.

[0324] The antigen binding portion according to the present disclosure may be or may comprise an antigen binding peptide / polypeptide or an antigen binding peptide / polypeptide complex. The antigen binding portion may comprise more than one peptide / polypeptide, which together form an antigen binding domain. The peptides / polypeptides may be covalently or non-covalently associated. In some embodiments, the peptides / polypeptides form part of a larger polypeptide comprising the peptides / polypeptides (e.g., in the case of a scFv portion comprising a VH region and a VL region, or in the case of a scFab comprising a VH-CH1 and a VL-CL).

[0325] In some embodiments, the antigen binding portion of the present invention comprises an antibody heavy chain variable (VH) region and an antibody light chain variable (VL) region of an antibody capable of binding to a given target antigen. In some embodiments, the antigen binding portion comprises an Fv portion formed by the VH region and VL region of an antibody capable of binding to a given target antigen or consists of it. In some embodiments, the VH region and the VL region can be provided in the same polypeptide and are joined by a linker sequence. In some embodiments, the antigen binding portion comprises or consists of a scFv portion that binds to a given target antigen.

[0326] The antigen-binding moieties of the present disclosure typically comprise six complementarity determining regions (CDRs); three in the heavy chain variable (VH) region: HC-CDR1, HC-CDR2, and HC-CDR3, and three in the light chain variable (VL) region: LC-CDR1, LC-CDR2, and LC-CDR3. Together, the six CDRs define the paratope of the antigen-binding moiety, which is the portion that binds to the target antigen.

[0327] The VH and VL regions contain framework regions (FRs) on both sides of each CDR, which provide a scaffold for the CDRs. From N-terminus to C-terminus, the VH region comprises the following structure: N-terminus-[HC-FR1]-[HC-CDR1]-[HC-FR2]-[HC-CDR2]-[HC-FR3]-[HC-CDR3]-[HC-FR4]-C-terminus; and the VL region comprises the following structure: N-terminus-[LC-FR1]-[LC-CDR1]-[LC-FR2]-[LC-CDR2]-[LC-FR3]-[LC-CDR3]-[LC-FR4]-C-terminus.

[0328] There are several different conventions for defining antibody CDRs and FRs, such as (i) the Kabat system, described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991); (ii) the Chothia system, described in Chothia et al., J. Mol. Biol. 196:901-917 (1987); and (iii) the international IMGT (ImMunoGeneTics) information system (LeFranc et al., Nucleic Acids Res. (2015) 43 (Database issue): D413-22), which uses the IMGT V-DOMAIN numbering convention as described in Lefranc et al., Dev. Comp. Immunol. (2003) 27:55-77.

[0329] The CDRs and FRs of the VH and VL regions of the antigen binding portions described herein are defined according to the Kabat system.

[0330] In some embodiments, the antigen binding portion comprises the CDRs of the antigen binding portion that binds to the variant Fc domain of the present disclosure. In some embodiments, the antigen binding portion comprises the FRs of the antigen binding portion that binds to the variant Fc domain of the present disclosure. In some embodiments, the antigen binding portion comprises the CDRs and FRs of the antigen binding portion that binds to the variant Fc domain of the present disclosure. That is, in some embodiments, the antigen binding portion comprises the VH region and VL region of the antigen binding portion that binds to the variant Fc domain of the present disclosure.

[0331] Wessels et al. Bioanal. (2017) 9(11): 849–59 describe the identification of an antibody that binds to an antibody comprising an Fc domain derived from human IgG1 comprising P329G, but does not bind to an antibody comprising an equivalent Fc domain lacking the P329G substitution. The antibody also binds to an antibody having an hIgG1-derived Fc region comprising P329G and further comprising L234A and L235A. Darowski et al., Protein Eng. Des. Sel. (2019) 32(5): 207-218 and Stock et al., Journal for ImmunoTherapy of Cancer (2022) 10: e005054 provide the structure of an anti-P329G Fab having an Fc region comprising P329G, L234A, and L235A. The anti-P329G Fab interacts with an Fc comprising P329G, L234A, and L235A in a 1: 1 stoichiometry. The epitope is disclosed as including positions N325 to P331 (including G329), and S267 to E272.

[0332] In some embodiments, the antigen binding portion comprises CDRs, FRs and / or VH and / or VL regions of an antigen binding molecule described herein that is bound to a variant Fc domain according to the present disclosure, or comprises CDRs, FRs and / or VH and / or VL regions derived from those regions of an antigen binding molecule described herein that is bound to a variant Fc domain according to the present disclosure. In some embodiments, the antigen binding molecule that is bound to a variant Fc domain according to the present disclosure is selected from the group consisting of: αP329G_VH1 / VL1, αP329G_VH2 / VL1, and αP329G_VH3 / VL1.

[0333] In some embodiments, the antigen binding portion comprises a VH region according to (1) or (2) below:

[0334] (1) A VH region comprising the following CDRs:

[0335] HC-CDR1 having the amino acid sequence of SEQ ID NO: 11

[0336] HC-CDR2 having the amino acid sequence of SEQ ID NO: 12

[0337] an HC-CDR3 having the amino acid sequence of SEQ ID NO: 13,

[0338] or a variant thereof, wherein 1 or 2 or 3 amino acids in HC-CDR1, and / or wherein 1 or 2 or 3 amino acids in HC-CDR2, and / or wherein 1 or 2 or 3 amino acids in HC-CDR3 are substituted with another amino acid.

[0339] (2) A VH region comprising the following CDRs:

[0340] HC-CDR1 having the amino acid sequence of SEQ ID NO: 11

[0341] HC-CDR2 having the amino acid sequence of SEQ ID NO: 19

[0342] an HC-CDR3 having the amino acid sequence of SEQ ID NO: 13,

[0343] or a variant thereof, wherein 1 or 2 or 3 amino acids in HC-CDR1, and / or wherein 1 or 2 or 3 amino acids in HC-CDR2, and / or wherein 1 or 2 or 3 amino acids in HC-CDR3 are substituted with another amino acid.

[0344] In some embodiments, the antigen binding portion comprises a VH region according to (3) or (4) below:

[0345] (3) A VH region comprising the following FRs:

[0346] HC-FR1 having the amino acid sequence of SEQ ID NO: 14

[0347] HC-FR2 having the amino acid sequence of SEQ ID NO: 15

[0348] HC-FR3 having the amino acid sequence of SEQ ID NO: 16

[0349] HC-FR4 having the amino acid sequence of SEQ ID NO: 17,

[0350] or a variant thereof, wherein 1 or 2 or 3 amino acids in HC-FR1, and / or wherein 1 or 2 or 3 amino acids in HC-FR2, and / or wherein 1 or 2 or 3 amino acids in HC-FR3, and / or wherein 1 or 2 or 3 amino acids in HC-FR4 are substituted with another amino acid.

[0351] (4) A VH region comprising the following FRs:

[0352] HC-FR1 having the amino acid sequence of SEQ ID NO: 21

[0353] HC-FR2 having the amino acid sequence of SEQ ID NO: 15

[0354] HC-FR3 having the amino acid sequence of SEQ ID NO: 22

[0355] HC-FR4 having the amino acid sequence of SEQ ID NO: 17,

[0356] or a variant thereof, wherein 1 or 2 or 3 amino acids in HC-FR1, and / or wherein 1 or 2 or 3 amino acids in HC-FR2, and / or wherein 1 or 2 or 3 amino acids in HC-FR3, and / or wherein 1 or 2 or 3 amino acids in HC-FR4 are substituted with another amino acid.

[0357] In some embodiments, the antigen binding portion comprises a VH region comprising CDRs according to (1) or (2) above and FRs according to (3) or (4) above.

[0358] In some embodiments, the antigen binding portion comprises a VH region according to (5) or (6) below:

[0359] (5) A VH region comprising the CDRs according to (1) and the FRs according to (3).

[0360] (6) A VH region comprising the CDRs according to (2) and the FRs according to (4).

[0361] (7) A VH region comprising the CDRs according to (2) and the FRs according to (3).

[0362] In some embodiments, the antigen binding portion comprises a VH region according to one of the following (8) to (10):

[0363] (8) a VH region comprising an amino acid sequence having at least 70% sequence identity (more preferably at least one of ≥75%, ≥80%, ≥85%, ≥86%, ≥87%, ≥88%, ≥89%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% sequence identity) to the amino acid sequence of SEQ ID NO: 10.

[0364] (9) a VH region comprising an amino acid sequence having at least 70% sequence identity (more preferably at least one of ≥75%, ≥80%, ≥85%, ≥86%, ≥87%, ≥88%, ≥89%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% sequence identity) to the amino acid sequence of SEQ ID NO: 18.

[0365] (10) a VH region comprising an amino acid sequence having at least 70% sequence identity (more preferably at least one of ≥75%, ≥80%, ≥85%, ≥86%, ≥87%, ≥88%, ≥89%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% sequence identity) to the amino acid sequence of SEQ ID NO: 20.

[0366] In some embodiments, the antigen binding portion comprises a VL region according to (11):

[0367] (11) A VL region comprising the following CDRs:

[0368] LC-CDR1 having the amino acid sequence of SEQ ID NO: 24

[0369] LC-CDR2 having the amino acid sequence of SEQ ID NO: 25

[0370] LC-CDR3 having the amino acid sequence of SEQ ID NO: 26,

[0371] or a variant thereof, wherein 1 or 2 or 3 amino acids in LC-CDR1, and / or wherein 1 or 2 or 3 amino acids in LC-CDR2, and / or wherein 1 or 2 or 3 amino acids in LC-CDR3 are substituted with another amino acid.

[0372] In some embodiments, the antigen binding portion comprises a VL region according to (12):

[0373] (12) A VL region comprising the following FRs:

[0374] LC-FR1 having the amino acid sequence of SEQ ID NO: 27

[0375] LC-FR2 having the amino acid sequence of SEQ ID NO: 28

[0376] LC-FR3 having the amino acid sequence of SEQ ID NO: 29

[0377] LC-FR4 having the amino acid sequence of SEQ ID NO: 30,

[0378] or a variant thereof, wherein 1 or 2 or 3 amino acids in LC-FR1, and / or wherein 1 or 2 or 3 amino acids in LC-FR2, and / or wherein 1 or 2 or 3 amino acids in LC-FR3, and / or wherein 1 or 2 or 3 amino acids in LC-FR4 are substituted with another amino acid.

[0379] In some embodiments, the antigen binding portion comprises a VL region according to (13):

[0380] (13) A VL region comprising the CDRs according to (11) and the FRs according to (12).

[0381] In some embodiments, the antigen binding portion comprises a VL region according to (14):

[0382] (14) a VL region comprising an amino acid sequence having at least 70% sequence identity (more preferably at least one of ≥75%, ≥80%, ≥85%, ≥86%, ≥87%, ≥88%, ≥89%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% sequence identity) to the amino acid sequence of SEQ ID NO: 23.

[0383] In some embodiments, the antigen binding portion comprises a VH region according to any one of (1) to (10) above and a VL region according to any one of (11) to (14) above.

[0384] In some embodiments, the components of the antigen binding portion comprise, or consist of, one or more polypeptides comprising a VH region comprising HC-CDR1, HC-CDR2, and HC-CDR3 as indicated in column A of Table A. In some embodiments, the components of the antigen binding portion comprise, or consist of, one or more polypeptides comprising a VL region comprising LC-CDR1, LC-CDR2, and LC-CDR3 as indicated in column B of Table A.

[0385] In some embodiments, the components of the antigen binding portion comprise or consist of one or more polypeptides comprising a VH region comprising HC-FR1, HC-FR2, HC-FR3, and HC-FR4 as indicated in column A of Table B. In some embodiments, the components of the antigen binding portion comprise or consist of one or more polypeptides comprising a VL region comprising LC-FR1, LC-FR2, LC-FR3, and LC-FR4 as indicated in column B of Table B.

[0386] In some embodiments, the components of the antigen binding portion comprise or consist of one or more polypeptides comprising an amino acid sequence having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%) amino acid sequence identity to the amino acid sequences indicated in column A of Table C. In some embodiments, the components of the antigen binding portion comprise one or more polypeptides comprising an amino acid sequence having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%) amino acid sequence identity to the amino acid sequences indicated in column B of Table C.

[0387] In some embodiments, the component of the antigen binding portion comprises or consists of an amino acid having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%) amino acid sequence identity to SEQ ID NO: 10, 18, or 20. In some embodiments, the component of the antigen binding portion comprises or consists of an amino acid having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%) amino acid sequence identity to SEQ ID NO: 23.

[0388] It will be understood that where components of an antigen binding moiety are provided in aspects and embodiments of the present disclosure, it is intended that the provided components are complementary and capable of associating to form a (complete, functional) antigen binding moiety.

[0389] Amino acid substitutions according to the present disclosure can be biochemically conservative. In some embodiments, if the amino acid to be substituted is provided in a row in rows 1 to 5 of the following table, the replacement amino acid is another different amino acid provided in the same row:

[0390]

[0391] For example, in some embodiments where the substitution is to a Met residue, the replacing amino acid can be selected from Ala, Val, Leu, He, Trp, Tyr, Phe, and norleucine.

[0392] In some embodiments, the replacement amino acid in the substitution may have the same side chain polarity as the amino acid residue it replaces. In some embodiments, the replacement amino acid in the substitution may have the same charge (at pH 7.4) as the amino acid residue it replaces:

[0393]

[0394]

[0395] That is, in some embodiments, a non-polar amino acid is replaced by another non-polar amino acid. In some embodiments, a polar amino acid is replaced by another polar amino acid. In some embodiments, an acidic polar amino acid is replaced by another acidic polar amino acid. In some embodiments, a basic polar amino acid is replaced by another basic polar amino acid. In some embodiments, a neutral amino acid is replaced by another neutral amino acid. In some embodiments, a positively charged amino acid is replaced by another positively charged amino acid. In some embodiments, a negatively charged amino acid is replaced by another negatively charged amino acid.

[0396] In some embodiments, the substitutions may be functionally conservative. That is, in some embodiments, the substitutions may not affect (or substantially not affect) one or more functional properties (e.g., target antigen binding) of the antigen-binding portion comprising the substitutions, compared to an equivalent unsubstituted molecule.

[0397] In some embodiments, the antigen binding portion of the present disclosure comprises a VH as described herein. In some embodiments, the antigen binding portion comprises a VL as described herein. In some embodiments, the antigen binding portion comprises one or more antibody heavy chain constant regions (CH). In some embodiments, the antigen binding portion comprises one or more antibody light chain constant regions (CL). In some embodiments, the antigen binding portion comprises a CH1, CH2, and / or CH3 region of an immunoglobulin (Ig). In some embodiments, the antigen binding portion comprises a linker sequence as described herein.

[0398] In some embodiments, the antigen-binding portion of the present disclosure comprises one or more polypeptides comprising: (i) a VH region comprising HC-CDR1, HC-CDR2, and HC-CDR3 as indicated in column A of Table A, and (ii) a VL region comprising LC-CDR1, LC-CDR2, and LC-CDR3 as indicated in column B of Table A, wherein the sequences of columns A and B are selected from the same row of Table A.

[0399] In some embodiments, the antigen binding portion of the present disclosure comprises one or more polypeptides comprising: (i) a VH region comprising an HC-CDR1 according to SEQ ID NO: 11, an HC-CDR2 according to SEQ ID NO: 19, and an HC-CDR3 according to SEQ ID NO: 13, and (ii) a VL region comprising an LC-CDR1 according to SEQ ID NO: 24, an LC-CDR2 according to SEQ ID NO: 25, and an LC-CDR3 according to SEQ ID NO: 26.

[0400] In some embodiments, the antigen binding portion of the present disclosure comprises one or more polypeptides comprising: (i) a VH region comprising HC-FR1, HC-FR2, HC-FR3, and HC-FR4 as indicated in column A of Table B, and (ii) a VL region comprising LC-FR1, LC-FR2, LC-FR3, and LC-FR4 as indicated in column B of Table B, wherein the sequences of columns A and B are selected from the same row of Table B.

[0401] In some embodiments, the antigen binding portion of the present disclosure comprises one or more polypeptides comprising: (i) a VH region comprising HC-FR1 according to SEQ ID NO:21, HC-FR2 according to SEQ ID NO:15, HC-FR3 according to SEQ ID NO:22, and HC-FR4 according to SEQ ID NO:17, and (ii) a VL region comprising LC-FR1 according to SEQ ID NO:27, LC-FR2 according to SEQ ID NO:28, LC-FR3 according to SEQ ID NO:29, and LC-FR4 according to SEQ ID NO:30.

[0402] In some embodiments, the antigen-binding portion of the present disclosure comprises one or more polypeptides comprising: (i) an amino acid sequence having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%) amino acid sequence identity to the amino acid sequence indicated in column A of Table C, and (ii) an amino acid sequence having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%) amino acid sequence identity to the amino acid sequence indicated in column B of Table C, wherein the sequences of columns A and B are selected from the same row of Table C.

[0403] In some embodiments, the antigen binding portions of the present disclosure comprise amino acids having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%) amino acid sequence identity to SEQ ID NO: 10, 18, or 20. In some embodiments, the antigen binding portions of the present disclosure comprise amino acids having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%) amino acid sequence identity to SEQ ID NO: 23.

[0404] In some embodiments, the antigen binding portion of the present disclosure comprises an amino acid having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%) amino acid sequence identity to SEQ ID NO: 20. In some embodiments, the antigen binding portion of the present disclosure comprises an amino acid having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%) amino acid sequence identity to SEQ ID NO: 23.

[0405] In some embodiments, the antigen binding portion of the present disclosure comprises or consists of an amino acid sequence that has at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%) amino acid sequence identity to SEQ ID NO: 33. In some embodiments, the antigen binding portion comprises or consists of an amino acid sequence that has at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%) amino acid sequence identity to SEQ ID NO: 34. In preferred embodiments, the antigen binding portion comprises or consists of amino acids having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) amino acid sequence identity to SEQ ID NO:35.

[0406] Chimeric Antigen Receptor (CAR)

[0407] In a specific embodiment, the membrane-anchored antigen binding (MAB) polypeptide is a chimeric antigen receptor (CAR).

[0408] The term "chimeric antigen receptor" or "chimeric receptor" or "CAR" refers to an antigen binding receptor composed of an extracellular portion of an antigen binding portion (e.g., a single-chain antibody domain) fused via a transmembrane domain and optionally additional spacer sequences to an intracellular signaling domain / co-signaling domain (such as, for example, CD3z and CD28).

[0409] In some aspects, the transmembrane domain comprises a portion of a murine / mouse or preferably human transmembrane domain. An example of such a transmembrane domain is the transmembrane domain of CD8, for example, having an amino acid sequence as shown in SEQ ID NO: 73 herein. In the context of the present invention, the transmembrane domain of a CAR may comprise or consist of an amino acid sequence as shown in SEQ ID NO: 73.

[0410] In another embodiment, the CAR provided herein may comprise the transmembrane domain of CD28, which is located in the human full-length CD28 protein as shown in SEQ ID NO: 86 (as shown by SEQ ID NO: to 179, 170 to 179, 171 to 179, 172 to 179, 173 to 179, 174 to 179, 175 to 179, 176 to 179, 177 to 179 or 178 to 179 of the present invention.

[0411] Alternatively, any protein having a transmembrane domain, as provided by the CD nomenclature, can be used as the transmembrane domain of the CAR protein used in accordance with the present invention.

[0412] In some embodiments, the transmembrane domain comprises a transmembrane domain of any one of the group consisting of CD27 (SEQ ID NO:81, encoded by SEQ ID NO:83), CD137 (SEQ ID NO:92, encoded by SEQ ID NO:91), OX40 (SEQ ID NO:96, encoded by SEQ ID NO:95), ICOS (SEQ ID NO:100, encoded by SEQ ID NO:99), DAP10 (SEQ ID NO:104, encoded by SEQ ID NO:103), DAP12 (SEQ ID NO:108, encoded by SEQ ID NO:107), CD3z (SEQ ID NO:113, encoded by SEQ ID NO:114), FCGR3A (SEQ ID NO:115, encoded by SEQ ID NO:116), NKG2D (SEQ ID NO:119, encoded by SEQ ID NO:120), CD8 (SEQ ID NO:129, encoded by SEQ ID NO:130), CD40 (SEQ ID NO:131), CD60 (SEQ ID NO:132), CD8 (SEQ ID NO:133), CD90 (SEQ ID NO:134), CD190 (SEQ ID NO:135), CD200 (SEQ ID NO:136), CD210 (SEQ ID NO:137), CD230 (SEQ ID NO:138), CD231 (SEQ ID NO:139), CD232 (SEQ ID NO:140), CD233 (SEQ ID NO:141), CD234 (SEQ ID NO:142), CD235 (SEQ ID NO:143), CD236 (SEQ ID NO:144), CD237 (SEQ ID NO:145), CD238 (SEQ ID NO:146), CD239 (SEQ ID NO:147), CD240 (SEQ ID NO:148), CD241 (SEQ ID NO:149), CD242 (SEQ ID NO:150), NO: 133, encoded by SEQ ID NO: 134), or a transmembrane fragment thereof that retains the ability to restrict CAR to the membrane.

[0413] Human sequences may be beneficial, for example, because (part of) the transmembrane domain may be accessible from the extracellular space and hence to the patient's immune system.In a preferred embodiment, the transmembrane domain comprises a human sequence. In such embodiments, the transmembrane domain comprises a transmembrane domain of any one of the group consisting of human CD27 (SEQ ID NO:82, encoded by SEQ ID NO:81), human CD137 (SEQ ID NO:90, encoded by SEQ ID NO:89), human OX40 (SEQ ID NO:94, encoded by SEQ ID NO:93), human ICOS (SEQ ID NO:98, encoded by SEQ ID NO:917), human DAP10 (SEQ ID NO:103, encoded by SEQ ID NO:102), human DAP12 (SEQ ID NO:106, encoded by SEQ ID NO:105), human CD3z (SEQ ID NO:111, encoded by SEQ ID NO:110), human FCGR3A (SEQ ID NO:113, encoded by SEQ ID NO:114), human NKG2D (SEQ ID NO:117, encoded by SEQ ID NO:118), human CD8 (SEQ ID NO:127, encoded by SEQ ID NO:129), human NKG2D (SEQ ID NO:128, encoded by SEQ ID NO:130), human NKG2D (SEQ ID NO:129, encoded by SEQ ID NO:131), human NKG2D (SEQ ID NO:129, encoded by SEQ ID NO:132), human NKG2D (SEQ ID NO:121, encoded by SEQ ID NO:133), human NKG2D (SEQ ID NO:123, encoded by SEQ ID NO:134), human NKG2D (SEQ ID NO:124, encoded by SEQ ID NO:135), human NKG2D (SEQ ID NO:125, encoded by SEQ ID NO:136), human NKG2D (SEQ ID NO:126, encoded by SEQ ID NO:137), human NKG2D (SEQ ID NO:127, encoded by SEQ ID NO:138), human NKG2D (SEQ ID NO: NO: 128), human CD40 (SEQ ID NO: 131, encoded by SEQ ID NO: 132), or a transmembrane fragment thereof that retains the ability to anchor the CAR to the membrane.

[0414] Preferably, the CAR used according to the present invention comprises at least one stimulatory signaling domain and / or at least one co-stimulatory signaling domain. Thus, the CAR provided herein preferably comprises a stimulatory signaling domain that provides T cell activation. The CAR provided herein may comprise a stimulatory signaling domain that is a fragment / polypeptide portion of murine / mouse or human CD3z (UniProt entry for human CD3z is P20963 (version 177, sequence number 2); UniProt entry for murine / mouse CD3z is P24161 (primary citable accession number) or Q9D3G3 (secondary citable accession number), version 143, sequence number 1), FCGR3A (UniProt entry for human FCGR3A is P08637 (version 178, sequence number 2)) or NKG2D (UniProt entry for human NKG2D is P26718 (version 151, sequence number 1); UniProt entry for murine / mouse NKG2D is O54709 (version 132, sequence number 2)).

[0415] Thus, the stimulatory signaling domain included in the CAR provided herein can be a fragment / polypeptide portion of full-length CD3z, FCGR3A or NKG2D. The amino acid sequence of mouse / mouse full-length CD3z or NKG2D is shown herein as SEQ ID NO: 111 (CD3z), 115 (FCGR3A) or 119 (NKG2D) (mouse / mouse such as encoded by the DNA sequence shown in SEQ ID NO: 112 (CD3z), 116 (FCGR3A) or 120 (NKG2D)). The amino acid sequence of human full-length CD3z, FCGR3A or NKG2D is shown herein as SEQ ID NO: 109 (CD3z), 113 (FCGR3A) or 117 (NKG2D) (human such as encoded by the DNA sequence shown in SEQ ID NO: 110 (CD3z), 114 (FCGR3A) or 118 (NKG2D)). The CAR used according to the present invention may comprise a fragment of CD3z, FCGR3A or NKG2D as a stimulatory domain, provided that it includes at least one signaling domain. In particular, any part / fragment of CD3z, FCGR3A or NKG2D is suitable as a stimulatory domain as long as it includes at least one signaling motif. However, more preferably, the CAR used according to the present invention comprises a polypeptide derived from human origin. Therefore, more preferably, the CAR provided herein comprises an amino acid sequence as shown herein as SEQ ID NO: 109 (CD3z), 113 (FCGR3A) or 117 (NKG2D) (human such as encoded by the DNA sequence shown by SEQ ID NO: 110 (CD3z), 114 (FCGR3A) or 118 (NKG2D)). In one embodiment, the CAR used according to the present invention may comprise or consist of an amino acid sequence as shown in SEQ ID NO: 146, SEQ ID NO: 149, SEQ ID NO: 151, or SEQ ID NO: 154. In another embodiment, the CAR comprises a sequence as set forth in SEQ ID NO: 151 or a sequence having up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 23, 24, 25, 26, 27, 28, 29 or 30 substitutions, deletions or insertions compared to SEQ ID NO: 151 and characterized by having stimulatory signaling activity. Specific configurations of CARs comprising a stimulatory signaling domain (SSD) are provided below and in the Examples and Figures. Stimulatory signaling activity can be determined; for example, by increased cytokine release measured by ELISA (IL-2, IFNγ, TNFα), increased proliferative activity (measured by increased cell number), or increased lytic activity measured by an LDH release assay.

[0416] In addition, the CAR provided herein preferably comprises at least one co-stimulatory signaling domain, which provides additional activity for T cells. The CAR provided herein may comprise a co-stimulatory signaling domain, which is mouse / mouse or human CD28 (the UniProt entry for human CD28 is P10747 (version number 173, sequence number 1); the UniProt entry for mouse / mouse CD28 is P31041 (version number 134, sequence number 2)), CD137 (the UniProt entry for human CD137 is Q07011 (version number 145, sequence number 1); the UniProt entry for mouse / mouse CD137 is P20334 (version number 139, sequence number 1) ), OX40 (the UniProt entry for human OX40 is P23510 (version 138, sequence number 1); the UniProt entry for rat / mouse OX40 is P43488 (version 119, sequence number 1)), ICOS (the UniProt entry for human ICOS is Q9Y6W8 (version 126, sequence number 1); the UniProt entry for rat / mouse ICOS is Q9WV40 (primary citable accession number) or Q9JL17 (secondary citable accession number), version 102, sequence number 2), CD27 ( The UniProt entry for human CD27 is P26842 (version 160, sequence number 2); the UniProt entry for mouse / mouse CD27 is P41272 (version 137, sequence number 1)), 4-1-BB (the UniProt entry for mouse / mouse 4-1-BB is P20334 (version 140, sequence number 1); the UniProt entry for human 4-1-BB is Q07011 (version 146, sequence number)), DAP10 (the UniProt entry for human DAP10 is Q9UBJ5 (version 25, SEQ ID NO: 1); the UniProt entry for mouse / mouse DAP10 is Q9QUJ0 (primary citable accession number) or Q9R1E7 (secondary citable accession number), version 101, SEQ ID NO: 1) or DAP12 (the UniProt entry for human DAP12 is O43914 (version 146, SEQ ID NO: 1); the UniProt entry for mouse / mouse DAP12 is O054885 (primary citable accession number) or Q9R1E7 (secondary citable accession number), version 123, SEQ ID NO: 1) fragment / polypeptide portion. The CAR provided herein may comprise a costimulatory signaling domain, which is a fragment / polypeptide portion of human or mouse / mouse CD40 (SEQ ID NO: 131, 133). In certain embodiments of the present invention, the CAR may comprise one or more (i.e., 1, 2, 3, 4, 5, 6, or 7) costimulatory signaling domains as defined herein.Thus, in the context of the present invention, the CAR may comprise a fragment / polypeptide portion of murine / mouse or preferably human CD137 as a first co-stimulatory signaling domain, and the second co-stimulatory signaling domain is selected from the group consisting of murine / mouse or preferably human CD27, CD28, CD137, OX40, ICOS, DAP10 and DAP12 or a fragment thereof. Preferably, the CAR comprises a co-stimulatory signaling domain derived from human origin. Thus, more preferably, the one or more co-stimulatory signaling domains contained in the CAR used according to the present invention may comprise or consist of an amino acid sequence as shown in SEQ ID NO: 74 or 76.

[0417] Thus, the costimulatory signaling domain that can be included in the CARs provided herein is a fragment / polypeptide portion of full-length CD27, CD28, CD137, OX40, ICOS, DAP10, DAP12, or CD40. The amino acid sequences of murine / mouse full-length CD27, CD28, CD137, OX40, ICOS, CD27, DAP10, DAP12, and CD40 are set forth herein as SEQ ID NO: 82 (CD27), 89 (CD28), 93 (CD137), 97 (OX40), 101 (ICOS), 105 (DAP10), 109 (DAP12), or 133 (CD40) (murine / mouse as encoded by the DNA sequences set forth in SEQ ID NO: 83 (CD27), 87 (CD28), 91 (CD137), 95 (OX40), 99 (ICOS), 103 (DAP10), 107 (DAP12), 134 (CD40)). However, since human sequences are most preferred in the context of the present invention, the co-stimulatory signaling domain that can optionally be included in the CAR proteins provided herein is a fragment / polypeptide portion of human full-length CD27, CD28, CD137, OX40, ICOS, DAP10, DAP12 or CD40. The amino acid sequence of human full-length CD27, CD28, CD137, OX40, ICOS, DAP10, DAP12 or CD40 is shown herein as SEQ ID NO: 82 (CD27), 86 (CD28), 90 (CD137), 94 (OX40), 98 (ICOS), 102 (DAP10), 106 (DAP12), 131 (CD40) (human as encoded by the DNA sequence set forth in SEQ ID NO: 81 (CD27), 85 (CD28), 89 (CD137), 93 (OX40), 97 (ICOS), 101 (DAP10), 105 (DAP12), 132 (CD40)).

[0418] In a preferred embodiment, the CAR comprises CD28 or a fragment thereof capable of activating T cells as a co-stimulatory signaling domain. The CAR provided herein may comprise a fragment of CD28 as a co-stimulatory signaling domain, provided that it comprises at least one signaling domain of CD28. In particular, any part / fragment of CD28 is suitable for use in the CAR according to the present invention as long as it comprises at least one signaling motif of CD28. The co-stimulatory signaling domains PYAP (AA 208 to 211 of CD28) and YMNM (AA 191 to 194 of CD28) are beneficial to the function of the CD28 polypeptide and the functional effects listed above. The amino acid sequence of the YMNM domain is shown in SEQ ID NO: 97; the amino acid sequence of the PYAP domain is shown in SEQ ID NO: 98. Therefore, in the CAR used according to the present invention, the CD28 polypeptide preferably comprises a sequence derived from the intracellular domain of the CD28 polypeptide having the sequence YMNM (SEQ ID NO: 121) and / or PYAP (SEQ ID NO: 122). In other embodiments, one or both of these domains are mutated to FMNM (SEQ ID NO: 123) and / or AYAA (SEQ ID NO: 124). Any of these mutations reduces the ability of transduced cells comprising CAR to release cytokines without affecting their proliferative capacity, and can be advantageously used to prolong viability and thus extend the therapeutic potential of transduced cells. Or, in other words, this non-functional mutation preferably enhances the persistence of cells transduced with CAR provided herein in vivo. However, these signal transduction motives can be present at any site within the intracellular domain of CAR provided herein.

[0419] In another preferred embodiment, CAR comprises CD137 or a fragment thereof capable of activating T cells as a co-stimulatory signaling domain. The CAR provided herein may comprise a fragment of CD137 as a co-stimulatory signaling domain, provided that at least one signaling domain of CD137 is included. In particular, as long as at least one signaling motif of CD137 is included, any part / fragment of CD137 is suitable for use in accordance with the present invention. In a preferred embodiment, the CD137 polypeptide contained in the CAR protein used according to the present invention comprises or consists of the amino acid sequence shown in SEQ ID NO: 76.

[0420] The specific configuration of CAR comprising a costimulatory signaling domain (CSD) is provided below and in the examples and drawings. Costimulatory signaling activity can be determined; for example, the cytokine release measured by ELISA (IL-2, IFNγ, TNFα) increases, the proliferation activity increases (measured by the increased number of cells), or the lytic activity measured by LDH release assay increases. As described above, in one embodiment of the present invention, the costimulatory signaling domain of CAR can be derived from human CD28 and / or CD137 genes or fragments thereof that can achieve T cell activation, defined as cytokine production, proliferation and lytic activity of T cells. CD28 and / or CD137 activity can be measured by ELISA release of cytokines or cytokine flow cytometry (such as interferon-γ (IFN-γ) or interleukin 2 (IL-2)), T cell proliferation measurement, for example by ki67 measurement, cell quantification by flow cytometry, or assessment of lytic activity by real-time impedance measurement of target cells (by using, for example, an ICELLligence instrument, as described in, for example, Thakur et al., Biosens Bioelectron. 35(1) (2012), 503-506; Krutzik et al., Methods Mol Biol. 699 (2011), 179-202; Ekkens et al., Infect Immun. 75(5) (2007), 2291-2296; Ge et al., Proc Natl Acad Sci US A. 99(5) (2002), 2983-2988; Düwell et al., Cell Death Differ.21(12)(2014),1825-1837, errata: Cell Death Differ.21(12)(2014),161).

[0421] CAR provided herein may include at least one linker (or "spacer"). The linker is typically a peptide with a length of up to 20 amino acids. Therefore, in the context of the present invention, the length of the linker may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids. For example, CAR provided herein may include a linker located between an extracellular domain, a transmembrane domain, a costimulatory signaling domain and / or a stimulating signaling domain comprising at least one antigen binding portion that can specifically bind to a mutated Fc domain. In addition, CAR provided herein may include a linker in an antigen binding portion, particularly between the immunoglobulin domains of the antigen binding portion (such as between the VH domain and the VL domain of an scFv). The advantage of such linkers is that they increase the possibility that different polypeptides of CAR (i.e., the extracellular domain, transmembrane domain, costimulatory signaling domain and / or stimulating signaling domain comprising at least one antigen binding portion) are independently folded and operate as expected. Thus, in the context of the present invention, the extracellular domain, transmembrane domain, costimulatory signaling domain and stimulatory signaling domain comprising at least one antigen binding portion can be contained in a single-chain multifunctional polypeptide. A single-chain fusion construct can, for example, be composed of one or more polypeptides comprising one or more extracellular domains, one or more transmembrane domains, one or more costimulatory signaling domains and / or one or more stimulatory signaling domains containing at least one antigen binding portion. Thus, the antigen binding portion, transmembrane domain, costimulatory signaling domain and stimulatory signaling domain can be connected by one or more identical or different peptide linkers as described herein. For example, in the CAR provided herein, the linker between the extracellular domain comprising at least one antigen binding portion and the transmembrane domain can comprise or consist of an amino acid sequence as shown in SEQ ID NO: 78 and an amino acid sequence. In another embodiment, the linker between the antigen binding portion and the transmembrane domain comprises or consists of an amino acid sequence as shown in SEQ ID NO: 80. Thus, the transmembrane domain, costimulatory signaling domain and / or stimulatory signaling domain can be connected to each other by a peptide linker or alternatively by direct fusion of the domains.

[0422] In a preferred embodiment according to the present invention, the antigen binding portion is a single-chain variable fragment (scFv). ScFv is a fusion protein of the heavy chain variable domain (VH) and the light chain variable domain (VL) of an antibody, connected to a short linker peptide of 10 to about 25 amino acids. The linker is generally rich in glycine to obtain flexibility, and rich in serine or threonine to obtain solubility, and the N-terminus of VH can be connected to the C-terminus of VL, or vice versa. In a preferred embodiment, the linker connects the N-terminus of the VL domain to the C-terminus of the VH domain. For example, in CAR provided herein, the linker can have an amino group and an amino acid sequence as shown in SEQ ID NO: 77. ScFv antibodies, for example, are described in Houston, JS, Methods in Enzymol 203 (1991) 46-96).

[0423] In some embodiments according to the present invention, the antigen binding portion is a single-chain Fab fragment or scFab, which is a polypeptide consisting of a heavy chain variable domain (VH), an antibody constant domain 1 (CH1), an antibody light chain variable domain (VL), an antibody light chain constant domain (CL), and a linker, wherein the antibody domain and the linker have one of the following orders in the N-terminal to C-terminal direction: a) VH-CH1-linker-VL-CL, b) VL-CL-linker-VH-CH1, c) VH-CL-linker-VL-CH1, or d) VL-CH1-linker-VH-CL; and wherein the linker is a polypeptide of at least 30 amino acids, preferably 32 to 50 amino acids. The single-chain Fab fragment is stabilized via a natural disulfide bond between the CL domain and the CH1 domain.

[0424] CAR provided herein or its portion can include a signal peptide.Such signal peptides will bring the protein to the surface of the T cell membrane.For example, in CAR provided herein, the signal peptide can have an amino group and an amino acid sequence as shown in SEQ ID NO:125 (as encoded by the DNA sequence shown in SEQ ID NO:126).

[0425] The components of the CARs described herein can be fused to each other in a variety of configurations to generate T cell activating CARs.

[0426] In some embodiments, CAR includes an extracellular domain consisting of a heavy chain variable domain (VH) and a light chain variable domain (VL) connected to a transmembrane domain. In a preferred embodiment, the VH domain is optionally fused to the N-terminus of the VL domain at the C-terminus via a peptide linker. In other embodiments, CAR further includes a stimulation signaling domain and / or a costimulatory signaling domain. In a specific embodiment of this type, CAR is essentially composed of a VH domain and a VL domain, a transmembrane domain, and a stimulation signaling domain optionally connected by one or more peptide linkers, wherein the VH domain is fused to the N-terminus of the VL domain at the C-terminus, and the VL domain is fused to the N-terminus of the transmembrane domain at the C-terminus, wherein the transmembrane domain is fused to the N-terminus of the stimulation signaling domain at the C-terminus. Optionally, CAR further includes a costimulatory signaling domain. In one such embodiment, the CAR consists essentially of a VH domain and a VL domain, a transmembrane domain, and a stimulatory signaling domain and a costimulatory signaling domain connected by one or more peptide linkers, wherein the VH domain is fused at the C-terminus to the N-terminus of the VL domain, and the VL domain is fused at the C-terminus to the N-terminus of the transmembrane domain, wherein the transmembrane domain is fused at the C-terminus to the N-terminus of the stimulatory signaling domain, wherein the stimulatory signaling domain is fused at the C-terminus to the N-terminus of the costimulatory signaling domain. In an alternative embodiment, the costimulatory signaling domain is connected to the transmembrane domain instead of the stimulatory signaling domain. In a preferred embodiment, the CAR consists essentially of a VH domain and a VL domain, a transmembrane domain, and a costimulatory signaling domain and a stimulatory signaling domain connected by one or more peptide linkers, wherein the VH domain is fused at the C-terminus to the N-terminus of the VL domain, and the VL domain is fused at the C-terminus to the N-terminus of the transmembrane domain, wherein the transmembrane domain is fused at the C-terminus to the N-terminus of the costimulatory signaling domain, and wherein the costimulatory signaling domain is fused at the C-terminus to the N-terminus of the stimulatory signaling domain.

[0427] The antigen binding portion, transmembrane domain, stimulatory signaling and / or co-stimulatory signaling domain can be fused to each other directly or through one or more peptide linkers comprising one or more amino acids, typically about 2 to 20 amino acids. Peptide linkers are known in the art and described herein. Suitable non-immunogenic peptide linkers include, for example, (G4S) n 、(SG4) n 、(G4S) n or G4 (SG4) nA peptide linker, wherein "n" is typically a number between 1 and 10, typically between 2 and 4. A preferred peptide linker for connecting the antigen-binding portion and the transmembrane portion is GGGGS (G4S) according to SEQ ID NO 78. Another preferred peptide linker for connecting the antigen-binding portion and the transmembrane portion is KPTTTPAPRPPTPAPTIASQPLSLRPEACRPAA GGAVHTRGLDFACD (CD8 stem) according to SEQ ID NO 80. An exemplary peptide linker suitable for connecting the variable heavy chain domain (VH) and the variable light chain domain (VL) is GGGSGGGSGGGSGGGS (G4S) according to SEQ ID NO 77.

[0428] In addition, the linker may comprise (a portion of) an immunoglobulin hinge region. In particular, when the antigen binding portion is fused to the N-terminus of the transmembrane domain, the fusion may be via an immunoglobulin hinge region or a portion thereof, with or without an additional peptide linker.

[0429] As described herein, the CAR used according to the present invention comprises an extracellular domain comprising at least one antigen binding portion. A CAR having a single antigen binding portion capable of specifically binding to a target cell antigen is useful and preferred, particularly in cases where high expression of CAR is desired. In such cases, the presence of more than one antigen binding portion specific for a target cell antigen may limit the expression efficiency of CAR. However, in other cases, it will be advantageous to have a CAR comprising two or more antigen binding portions specific for a target cell antigen, such as to optimize targeting of a target site or to allow cross-linking of target cell antigens.

[0430] In a specific embodiment, the CAR comprises an antigen binding portion that is capable of specifically binding to a mutant Fc domain, particularly a variant CH2-CH3 region comprising the amino acid substitution P329G according to EU numbering. In one embodiment, the antigen binding portion that is capable of specifically binding to a variant CH2-CH3 region comprising the amino acid substitution P329G according to EU numbering is a scFv.

[0431] In one embodiment, the antigen binding portion is fused to the N-terminus of the transmembrane domain at the C-terminus of the scFv fragment, optionally fused by a peptide linker. In one embodiment, the peptide linker comprises the amino acid sequence KPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFAC D (SEQ ID NO: 80). In one embodiment, the transmembrane domain is a transmembrane domain, which is a transmembrane domain selected from the group consisting of CD8, CD4, CD3z, CD40, FCGR3A, NKG2D, CD27, CD28, CD137, OX40, ICOS, DAP10 or DAP12 transmembrane domain or a fragment thereof. In a preferred embodiment, the transmembrane domain is a CD8 transmembrane domain or a fragment thereof. In a specific embodiment, the transmembrane domain comprises the amino acid sequence of IYIWAPLAGTCGVLLLSLVIT (SEQ ID NO: 73) or consists of. In one embodiment, CAR further comprises a costimulatory signaling domain (CSD). In one embodiment, the transmembrane domain of the CAR is fused to the N-terminus of the costimulatory signaling domain at the C-terminus. In one embodiment, the costimulatory signaling domain is individually selected from the group consisting of: the intracellular domains of CD27, CD28, CD137, OX40, ICOS, DAP10, and DAP12, or fragments thereof, as described above. In a preferred embodiment, the costimulatory signaling domain is the intracellular domain of CD28 or a fragment thereof. In a preferred embodiment, the costimulatory signaling domain comprises the intracellular domain of CD28 or a fragment thereof that retains CD28 signaling. In another preferred embodiment, the costimulatory signaling domain comprises the intracellular domain of CD137 or a fragment thereof that retains CD137 signaling. In a specific embodiment, the costimulatory signaling domain comprises SEQ ID NO: 74 or consists thereof. In another specific embodiment, the costimulatory signaling domain comprises SEQ ID NO: 76 or consists thereof. In one embodiment, the CAR further comprises a stimulatory signaling domain. In one embodiment, the costimulatory signaling domain of the CAR is fused to the N-terminus of the stimulatory signaling domain at the C-terminus. In one embodiment, at least one stimulatory signaling domain is individually selected from the intracellular domain of the group consisting of CD3z, FCGR3A and NKG2D or a fragment thereof. In a preferred embodiment, the costimulatory signaling domain is the intracellular domain of CD3z or a fragment thereof that retains CD3z signaling. In a specific embodiment, the costimulatory signaling domain comprises SEQ ID NO: 75 or consists thereof.

[0432] In one embodiment, CAR is fused to a reporter protein, particularly fused to GFP or an enhanced analog thereof. In one embodiment, CAR is fused to the N-terminus of eGFP (enhanced green fluorescent protein) at the C-terminus, optionally fused via a peptide linker as described herein. In a preferred embodiment, the peptide linker is GEGRGSLLTCGDVEENPGP (T2A) according to SEQ ID NO: 79.

[0433] In a specific embodiment, CAR comprises a transmembrane domain and an extracellular domain comprising at least one antigen binding portion, wherein the at least one antigen binding portion is a scFv capable of specifically binding to a variant CH2-CH3 region comprising an amino acid substitution P329G according to EU numbering. The P329G mutation reduces Fcγ receptor binding. In one embodiment, CAR comprises a transmembrane domain (TD), a costimulatory signaling domain (CSD) and a stimulatory signaling domain (SSD). In such an embodiment, CAR has a configuration scFv-TD-CSD-SSD. In a preferred embodiment, CAR has a configuration VH-VL-TD-CSD-SSD. In a more specific such embodiment, CAR has a configuration VH-linker-VL-linker-TD-CSD-SSD.

[0434] In a specific embodiment, the antigen binding portion is a scFv capable of specifically binding to a variant CH2-CH3 region comprising the amino acid substitution P329G according to EU numbering, wherein the antigen binding portion comprises at least one heavy chain complementarity determining region (CDR) selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13 and at least one light chain CDR selected from SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26.

[0435] In another specific embodiment, the antigen binding portion is a scFv capable of specifically binding to a variant CH2-CH3 region comprising the amino acid substitution P329G according to EU numbering, wherein the antigen binding portion comprises at least one heavy chain complementarity determining region (CDR) selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 19 and SEQ ID NO: 13 and at least one light chain CDR selected from SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26.

[0436] In one embodiment, a CAR is provided that is capable of specifically binding to a variant CH2-CH3 region comprising the amino acid substitution P329G according to EU numbering, wherein the CAR comprises, in order from N-terminus to C-terminus:

[0437] (i) a heavy chain variable domain (VH) comprising a heavy chain complementarity determining region (CDR) 1 of SEQ ID NO: 11, a heavy chain CDR 2 of SEQ ID NO: 12, and a heavy chain CDR 3 of SEQ ID NO: 13,

[0438] (ii) a peptide linker, in particular a peptide linker of SEQ ID NO: 77,

[0439] (iii) a light chain variable domain (VL) comprising light chain CDR 1 of SEQ ID NO: 24, light chain CDR 2 of SEQ ID NO: 25, and light chain CDR 3 of SEQ ID NO: 26,

[0440] (iv) a peptide linker, in particular a peptide linker of SEQ ID NO: 80,

[0441] (v) a transmembrane domain, in particular the transmembrane domain of SEQ ID NO: 73,

[0442] (vi) a costimulatory signaling domain, in particular a costimulatory signaling domain of SEQ ID NO: 74 or 76, and

[0443] (vii) a stimulatory signaling domain, in particular a stimulatory signaling domain of SEQ ID NO: 75.

[0444] In one embodiment, a CAR is provided that is capable of specifically binding to a variant CH2-CH3 region comprising the amino acid substitution P329G according to EU numbering, wherein the CAR comprises, in order from N-terminus to C-terminus:

[0445] (i) a heavy chain variable domain (VH) comprising a heavy chain complementarity determining region (CDR) 1 of SEQ ID NO: 11, a heavy chain CDR 2 of SEQ ID NO: 19, and a heavy chain CDR 3 of SEQ ID NO: 13,

[0446] (ii) a peptide linker, in particular a peptide linker of SEQ ID NO: 77,

[0447] (iii) a light chain variable domain (VL) comprising light chain CDR 1 of SEQ ID NO: 24, light chain CDR 2 of SEQ ID NO: 25, and light chain CDR 3 of SEQ ID NO: 26,

[0448] (iv) a peptide linker, in particular a peptide linker of SEQ ID NO: 80,

[0449] (v) a transmembrane domain, in particular the transmembrane domain of SEQ ID NO: 73,

[0450] (vi) a costimulatory signaling domain, in particular a costimulatory signaling domain of SEQ ID NO: 74 or 76, and

[0451] (vii) a stimulatory signaling domain, in particular a stimulatory signaling domain of SEQ ID NO: 75.

[0452] In one embodiment, a CAR is provided that is capable of specifically binding to a variant CH2-CH3 region comprising the amino acid substitution P329G according to EU numbering, wherein the CAR comprises, in order from N-terminus to C-terminus:

[0453] (i) a heavy chain variable domain (VH),

[0454] (ii) a peptide linker, in particular a peptide linker of SEQ ID NO: 77,

[0455] (iii) a light chain variable domain (VL) that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 23,

[0456] wherein the VH domain and the VL domain are capable of forming an antigen-binding portion that binds to an Fc domain comprising the amino acid mutation P329G according to EU numbering,

[0457] (iv) a peptide linker, in particular a peptide linker of SEQ ID NO: 80,

[0458] (v) a transmembrane domain, in particular a transmembrane domain that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 73,

[0459] (vi) a costimulatory signaling domain, in particular a costimulatory signaling domain that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 74 or 76, and

[0460] (vii) a stimulatory signaling domain, particularly a stimulatory signaling domain that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:75.

[0461] In one embodiment, a CAR is provided that is capable of specifically binding to a variant CH2-CH3 region comprising the amino acid substitution P329G according to EU numbering, wherein the CAR comprises, in order from N-terminus to C-terminus:

[0462] (i) a heavy chain variable domain (VH) that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 10,

[0463] (ii) a peptide linker, in particular a peptide linker of SEQ ID NO: 77,

[0464] (iii) a light chain variable domain (VL) that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 23,

[0465] (iv) a peptide linker, in particular a peptide linker of SEQ ID NO: 80,

[0466] (v) a transmembrane domain, in particular a transmembrane domain that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 73,

[0467] (vi) a costimulatory signaling domain, in particular a costimulatory signaling domain that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 74 or 76, and

[0468] (vii) a stimulatory signaling domain, particularly a stimulatory signaling domain that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:75.

[0469] In one embodiment, a CAR is provided that is capable of specifically binding to a variant CH2-CH3 region comprising the amino acid substitution P329G according to EU numbering, wherein the CAR comprises, in order from N-terminus to C-terminus:

[0470] (i) a heavy chain variable domain (VH) that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 18,

[0471] (ii) a peptide linker, in particular a peptide linker of SEQ ID NO: 77,

[0472] (iii) a light chain variable domain (VL) that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 23,

[0473] (iv) a peptide linker, in particular a peptide linker of SEQ ID NO: 80,

[0474] (v) a transmembrane domain, in particular a transmembrane domain that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 73,

[0475] (vi) a costimulatory signaling domain, in particular a costimulatory signaling domain that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 74 or 76, and

[0476] (vii) a stimulatory signaling domain, particularly a stimulatory signaling domain that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:75.

[0477] In one embodiment, a CAR is provided that is capable of specifically binding to a variant CH2-CH3 region comprising the amino acid substitution P329G according to EU numbering, wherein the CAR comprises, in order from N-terminus to C-terminus:

[0478] (i) a heavy chain variable domain (VH) that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 20,

[0479] (ii) a peptide linker, in particular a peptide linker of SEQ ID NO: 77,

[0480] (iii) a light chain variable domain (VL) that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 23,

[0481] (iv) a peptide linker, in particular a peptide linker of SEQ ID NO: 80,

[0482] (v) a transmembrane domain, in particular a transmembrane domain that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 73,

[0483] (vi) a costimulatory signaling domain, in particular a costimulatory signaling domain that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 76, and

[0484] (vii) a stimulatory signaling domain, particularly a stimulatory signaling domain that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:75.

[0485] In one embodiment, a CAR is provided that is capable of specifically binding to a variant CH2-CH3 region comprising the amino acid substitution P329G according to EU numbering, wherein the CAR comprises, in order from N-terminus to C-terminus:

[0486] (i) a heavy chain variable domain (VH) that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 20,

[0487] (ii) a peptide linker, in particular a peptide linker of SEQ ID NO: 77,

[0488] (iii) a light chain variable domain (VL) that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 23,

[0489] (iv) a peptide linker, in particular a peptide linker of SEQ ID NO: 80,

[0490] (v) a transmembrane domain, in particular a transmembrane domain that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 73,

[0491] (vi) a costimulatory signaling domain, particularly a costimulatory signaling domain that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 74, and

[0492] (vii) a stimulatory signaling domain, particularly a stimulatory signaling domain that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:75.

[0493] In one embodiment, a CAR is provided that is capable of specifically binding to a variant CH2-CH3 region comprising the amino acid substitution P329G according to EU numbering and is capable of activating T cells, wherein the CAR comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the following amino acid sequence: SEQ ID NO: 146. In one embodiment, a CAR is provided that comprises the following amino acid sequence: SEQ ID NO: 146.

[0494] In one embodiment, a CAR is provided that is capable of specifically binding to a variant CH2-CH3 region comprising the amino acid substitution P329G according to EU numbering and is capable of activating T cells, wherein the CAR comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the following amino acid sequence: SEQ ID NO: 149. In one embodiment, a CAR is provided that comprises the following amino acid sequence: SEQ ID NO: 149.

[0495] In one embodiment, a CAR is provided that is capable of specifically binding to a variant CH2-CH3 region comprising the amino acid substitution P329G according to EU numbering and capable of activating T cells, wherein the CAR comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the following amino acid sequence: SEQ ID NO: 151. In one embodiment, a CAR is provided that comprises the following amino acid sequence: SEQ ID NO: 151.

[0496] In one embodiment, a CAR is provided that is capable of specifically binding to a variant CH2-CH3 region comprising the amino acid substitution P329G according to EU numbering and capable of activating T cells, wherein the CAR comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the following amino acid sequence: SEQ ID NO: 154. In one embodiment, a CAR is provided that comprises the following amino acid sequence: SEQ ID NO: 154.

[0497] In one embodiment, CAR is fused to a reporter protein, particularly fused to GFP or an enhanced analog thereof. In one embodiment, CAR is fused to the N-terminus of eGFP (enhanced green fluorescent protein) at the C-terminus, optionally fused via a peptide linker as described herein. In a preferred embodiment, the peptide linker is SEQ ID NO: 79's GEGRGSLLTCGDVEENPGP (T2A).

[0498] Recombinant CD3-TCR complex

[0499] In a specific embodiment, the membrane-anchored antigen binding (MAB) polypeptide comprises at least one recombinant CD3-TCR complex polypeptide.

[0500] The CD3-TCR complex (sometimes also referred to as the TCR-CD3 complex, see, e.g., Dong et al., Nature

[0501] (2019) 573(7775):546-552) is a polypeptide complex expressed on the cell surface of T cells that is involved in antigen-specific T cell activation. The structure and function of the CD3-TCR complex have been reviewed, for example, in Mariuzza et al., J Biol Chem. 2020 Jan 24; 295(4):914–925, the entire contents of which are incorporated herein by reference.

[0502] In mammals, the CD3-TCR complex contains a variety of TCR polypeptides that together form a heterodimeric TCR (TCRα and TCRβ, or TCRy and TCRδ) for antigen recognition, provided in a non-covalent association with the invariant CD3ε, CD3δ, CD3γ, and CD3ζ polypeptides. The classic octameric CD3-TCR complex contains a heterodimer of TCRα and TCRβ (ie, TCRαβ) or a heterodimer of TCRy and TCRδ (ie, TCRγδ), a heterodimer comprising CD3ε and CD3δ (ie, CD3δε), a heterodimer comprising CD3ε and CD3γ (ie, CD3γε), and a CD3ζ homodimer (ie, CD3ζζ). Such TCR-CD3 complexes can be expressed as CD3γε / CD3δε / CD3ζζ / TCRαβ and CD3γε / CD3δε / CD3ζζ / TCRγδ, respectively (see, e.g., Zheng et al., Nature (2019) 573(7775):546–552).

[0503] In some embodiments, the CD3-TCR complex is a CD3-TCRα / β complex. In some embodiments, the CD3-TCR complex is a CD3-TCRy / δ complex. The CD3-TCRα / β complex may comprise TCRα and TCRβ polypeptides, and further comprise CD3γ, CD3ε, CD3δ and / or CD3ζ polypeptides. The CD3-TCRα / β complex may comprise CD3γε / CD3δε / CD3ζζ / TCRαβ or consist thereof. The CD3-TCRγ / δ complex may comprise TCRγ and TCRδ polypeptides, and further comprise CD3γ, CD3ε, CD3δ and / or CD3ζ polypeptides. The CD3-TCRγ / δ complex may comprise CD3γε / CD3δε / CD3ζζ / TCRγ / δ or consist thereof.

[0504] Herein, "CD3-TCR complex polypeptide" refers to a constituent polypeptide of the CD3-TCR complex. In some embodiments, the CD3-TCR complex polypeptide is selected from TCRα, TCRβ, TCRγ, TCRδ, TRAC, TRBC1, TRBC2, TRGC1, TRGC2, TRDC, CD3ε, CD3δ, CD3γ, CD3ζ, and CD3η. In some embodiments, the CD3-TCR complex polypeptide is a recombinant CD3-TCR complex polypeptide as described herein.

[0505] In this specification, "TCRα", "TCRβ", "TCRγ", "TCRδ", "TRAC", "TRBC1", "TRBC2", "TRGC1", "TRGC2", "TRDC", "CD3ε", "CD3δ", "CD3γ", "CD3ζ" and "CD3η" refer to TCRα, TCRβ, TCRγ, TCRδ, TRAC, TRBC1, TRBC2, TRGC1, TRGC2, TRDC, CD3ε, CD3δ, CD3γ, CD3ζ and CD3η from any species, respectively, and include isoforms, fragments, variants or homologs from any species. In some embodiments, TCRα, TCRβ, TCRγ, TCRδ, TRAC, TRBC1, TRBC2, TRGC1, TRGC2, TRDC, CD3ε, CD3δ, CD3γ, CD3ζ and CD3η from any species, respectively. AC, TRBC1, TRBC2, TRGC1, TRGC2, TRDC, CD3ε, CD3δ, CD3γ, CD3ζ or CD3η are from a mammal (e.g., therian, placental, epitherian, preptotheria, archontan, primate (rhesus monkey, cynomolgus monkey, non-human primate or human). In some embodiments, TCRα, TCRβ, TCRγ, TCRδ, TRAC, TRBC1, TRBC2, TRGC1, TRGC2, TRDC, CD3ε, CD3δ, CD3γ, CD3ζ or CD3η are human.

[0506] As used herein, an isoform, fragment, variant or homolog of a given reference protein (e.g., TCRα, TCRβ, TCRγ, TCRδ, TRAC, TRBC1, TRBC2, TRGC1, TRGC2, TRDC, CD3ε, CD3δ, CD3γ, CD3ζ or CD3η) can be characterized by having at least 70% sequence identity, preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% amino acid sequence identity to the amino acid sequence of the reference protein.

[0507] A "fragment" generally refers to a portion of a reference protein. A "variant" generally refers to a protein having an amino acid sequence that comprises one or more amino acid substitutions, insertions, deletions, or other modifications relative to the amino acid sequence of a reference protein, but retains a substantial degree of sequence identity (e.g., at least 60%) with the amino acid sequence of the reference protein. An "isoform" generally refers to a variant of a reference protein expressed by the same species as the reference protein. A "homolog" generally refers to a variant of a reference protein produced by a different species than the reference protein. Homologs include orthologs.

[0508] Isoforms, fragments, variants or homologs of a given reference protein may optionally be characterized by having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) amino acid sequence identity to the amino acid sequence of a specified isoform of the related protein from a given species (e.g., human) in its immature or mature (i.e., after processing to remove the signal peptide) form.

[0509] In some embodiments, the TCRα comprises an amino acid sequence that has at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%) amino acid sequence identity to SEQ ID NO: 157. In some embodiments, the TRAC comprises or consists of an amino acid sequence that has at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%) amino acid sequence identity to SEQ ID NO: 157.

[0510] In some embodiments, the TCRβ comprises an amino acid sequence having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%) amino acid sequence identity to SEQ ID NO: 161 or 165. In some embodiments, TRBC1 comprises or consists of an amino acid sequence having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%) amino acid sequence identity to SEQ ID NO: 161. In some embodiments, TRBC2 comprises or consists of an amino acid sequence having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) amino acid sequence identity to SEQ ID NO: 165.

[0511] In some embodiments, TCRγ comprises an amino acid sequence having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%) amino acid sequence identity to SEQ ID NO: 169 or 173. In some embodiments, TRGC1 comprises or consists of an amino acid sequence having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%) amino acid sequence identity to SEQ ID NO: 169. In some embodiments, TRGC2 comprises or consists of an amino acid sequence having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) amino acid sequence identity to SEQ ID NO: 173.

[0512] In some embodiments, the TCRδ comprises an amino acid sequence having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%) amino acid sequence identity to SEQ ID NO: 177. In some embodiments, the TRDC comprises or consists of an amino acid sequence having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%) amino acid sequence identity to SEQ ID NO: 177.

[0513] In some embodiments, CD3ε comprises or consists of an amino acid sequence having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) amino acid sequence identity to SEQ ID NO: 181 or 186.

[0514] In some embodiments, CD3δ comprises or consists of an amino acid sequence having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%) amino acid sequence identity to SEQ ID NO: 187 or 192.

[0515] In some embodiments, CD3γ comprises or consists of an amino acid sequence having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%) amino acid sequence identity to SEQ ID NO: 193 or 198.

[0516] In some embodiments, CD3ζ comprises or consists of an amino acid sequence having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) amino acid sequence identity to SEQ ID NO: 199 or 204.

[0517] In some embodiments, CD3η comprises or consists of an amino acid sequence having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%) amino acid sequence identity to SEQ ID NO: 205 or 207.

[0518] In some embodiments, a recombinant CD3-TCR complex polypeptide according to the present disclosure comprises:

[0519] (i) an antigen binding portion or component thereof as described herein; and

[0520] (ii) a CD3-TCR complex association domain as described herein.

[0521] In some embodiments, in the amino acid sequence of a recombinant CD3-TCR complex polypeptide, the amino acid sequence of the antigen-binding portion / component thereof is located N-terminus to the amino acid sequence of the CD3-TCR complex associating domain. That is, in some embodiments, the recombinant CD3-TCR complex polypeptide comprises the following structure: N-terminus-[...]-[antigen-binding portion / component thereof]-[CD3-TCR complex associating domain]-[...]-C-terminus.

[0522] In some embodiments, the recombinant CD3-TCR complex polypeptides according to the present disclosure do not comprise a domain or amino acid sequence containing an immunoreceptor tyrosine-based activation motif (ITAM). The ITAM comprises an amino acid sequence according to YXXL / I (SEQ ID NO: 285), where "X" represents any amino acid. In proteins containing ITAMs, sequences according to YXXL / I are typically separated by 6 to 8 amino acids (i.e., they conform to the formula: YXXL / I(X) 6-8 YXXL / I; SEQ ID NO: 286). When a phosphate group is added to a tyrosine residue of an ITAM by a tyrosine kinase, a signaling cascade is initiated within the cell. ITAM-containing sequences include the intracellular domains of CD3ζ and FcγRI. In some embodiments, a recombinant CD3-TCR complex polypeptide according to the present disclosure does not comprise the amino acid sequence set forth in SEQ ID NO: 203. In some embodiments, a recombinant CD3-TCR complex polypeptide does not comprise the amino acid sequence according to SEQ ID NO: 286. In some embodiments, a recombinant CD3-TCR complex polypeptide does not comprise the amino acid sequence according to SEQ ID NO: 285.

[0523] In some embodiments, the recombinant CD3-TCR complex polypeptide according to the present disclosure does not include a costimulatory sequence. As mentioned herein, a "costimulatory sequence" refers to an amino acid sequence that provides costimulation of immune cells expressing recombinant CD3-TCR complex polypeptides. Costimulation promotes proliferation and survival, and can also promote cytokine production, differentiation, cytotoxic function and memory formation. The molecular mechanism of T cell costimulation is reviewed, for example, in Chen and Flies, (2013) Nat Rev Immunol 13 (4): 227-242. The costimulatory sequence can be or can be derived from the intracellular domain of a costimulatory protein. Costimulatory proteins include CD28, 4-1BB, ICOS, CD27, OX40, HVEM, CD2, SLAM, TIM-1, CD30, GITR, DR3, CD226 and LIGHT. In some embodiments, the recombinant CD3-TCR complex polypeptide according to the present disclosure does not include the amino acid sequence shown in SEQ ID NO:138 (the intracellular domain of human 4-1BB).

[0524] The recombinant CD3-TCR complex polypeptides of the present disclosure comprise a CD3-TCR complex association domain. The primary function of the CD3-TCR complex association domain is to provide for the formation of a polypeptide complex comprising a recombinant CD3-TCR complex polypeptide according to the present disclosure and one or more CD3-TCR complex polypeptides.

[0525] A "CD3-TCR complex association domain" refers to a domain through which a polypeptide comprising the domain is able to associate with a CD3-TCR complex polypeptide (e.g., a CD3-TCR complex polypeptide as described above). Thus, a CD3-TCR complex association domain according to the present disclosure comprises or consists of an amino acid sequence that confers the ability of a polypeptide comprising the domain to associate with a CD3-TCR complex polypeptide to form a polypeptide complex comprising the CD3-TCR complex polypeptide and a polypeptide carrying the CD3-TCR complex association domain.

[0526] The association between a CD3-TCR complex association domain / a polypeptide comprising a CD3-TCR complex association domain and a CD3-TCR complex polypeptide can be characterized by a non-covalent protein:protein interaction. In some embodiments, the association comprises electrostatic interactions (e.g., ionic bonds, hydrogen bonds) and / or van der Waals forces.

[0527] In some embodiments, the CD3-TCR complex association domain is or is derived from the amino acid sequence of a CD3-TCR complex polypeptide. It should be understood that the CD3-TCR complex association domain can be or can be derived from the region of the CD3-TCR complex polypeptide through which the CD3-TCR complex polypeptide interacts with other CD3-TCR complex polypeptides to form a polypeptide complex. In some embodiments, the CD3-TCR complex association domain is or is derived from the amino acid sequence of a region of the CD3-TCR complex polypeptide that is required for the association between the CD3-TCR complex polypeptide and other CD3-TCR complex polypeptides to form a polypeptide complex comprising such polypeptides.

[0528] The regions of the CD3-TCR complex polypeptides required for such interactions can be determined by site-directed mutagenesis and / or truncation studies, wherein the amino acid sequence of the CD3-TCR complex polypeptide is altered or truncated, and the effect of such alterations / truncations on the ability of the CD3-TCR complex polypeptide to bind to other CD3-TCR complex polypeptides is assessed. Suitable techniques for studying such protein:protein interactions include, for example, resonance energy transfer techniques, such as fluorescence resonance energy transfer (FRET) and bioluminescence resonance energy transfer (BRET), using appropriately labeled interaction partners, for example as described in Ciruela, Curr. Opin. Biotechnol. (2008) 19(4): 338-43.

[0529] As used herein, polypeptides, domains, and amino acid sequences that are "derived from" a reference polypeptide / domain / amino acid sequence have at least 60% (preferably one of ≥70%, ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%) amino acid sequence identity to the amino acid sequence of the reference polypeptide / domain / amino acid sequence. Polypeptides, domains, and amino acid sequences that are "derived from" a reference polypeptide / domain / amino acid sequence preferably retain the functional and / or structural properties of the reference polypeptide / domain / amino acid sequence.

[0530] In some embodiments, the CD3-TCR complex association domain comprises modifications that promote association with CD3-TCR complex polypeptides. In some embodiments, the CD3-TCR complex association domain comprises modifications that promote heteromerization (i.e., association with non-identical CD3-TCR complex polypeptides).

[0531] As used herein, "modification" refers to the difference relative to a reference amino acid sequence. The reference amino acid sequence can be an amino acid sequence encoded by the most common nucleotide sequence of a gene encoding the relevant protein. In the embodiments herein (and more generally in the art), "modification" may also be referred to as "substitution" or "mutation". Modification generally includes the replacement of an amino acid residue. The replacement of an amino acid residue includes replacing an amino acid residue with a different "replacement" amino acid residue. The replacement amino acid residue modified according to the present disclosure can be a naturally occurring amino acid residue (i.e., encoded by the genetic code) that is different from the amino acid residue at the relevant position of the amino acid sequence before modification, selected from the group consisting of: alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp), cysteine ​​(Cys), glutamine (Gln), glutamic acid (Glu), glycine (Gly), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), methionine (Met), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), tryptophan (Trp), tyrosine (Tyr), and valine (Val). In some embodiments, the replacement amino acid residue modified can be a non-naturally occurring amino acid residue, i.e., an amino acid residue other than the amino acid residues listed in the preceding sentence. Examples of non-naturally occurring amino acid residues include norleucine, ornithine, norvaline, homoserine, aib, and other amino acid residue analogs such as those described in Ellman et al., Meth. Enzym. 202 (1991) 301-336.

[0532] For example, in the examples herein, the CD3-TCR complex association domain derived from TRAC comprises a modification in which the threonine residue at position 47 (numbered relative to SEQ ID NO: 157) is replaced with a cysteine ​​residue (SEQ ID NO: 208), and the CD3-TCR complex association domain derived from TCRβ comprises a modification in which the serine residue at position 56 (numbered relative to SEQ ID NO: 209) is replaced with a cysteine ​​residue (SEQ ID NO: 161). The introduction of these cysteine ​​residues promotes heteromerization between the modified domains through the formation of interchain disulfide bridges.

[0533] Embodiments in which the CD3-TCR complex association domain further comprises modifications to promote association with a CD3-TCR complex polypeptide are contemplated to be particularly relevant to aspects and embodiments of the present disclosure in which recombinant CD3-TCR complex polypeptides comprising such a CD3-TCR complex association domain are provided for use with another recombinant CD3-TCR complex polypeptide. For example, such a CD3-TCR complex association domain is contemplated to be particularly useful in a first recombinant CD3-TCR complex polypeptide and / or a second recombinant CD3-TCR complex polypeptide of a polypeptide complex of the present disclosure comprising such a recombinant CD3-TCR complex polypeptide.

[0534] In some embodiments, the CD3-TCR complex association domain is or is derived from the CD3-TCR complex association domain of CD3ε. In some embodiments, the CD3-TCR complex association domain is or is derived from the region of CD3ε required for association with CD3γ and / or CD3δ (i.e., to form a CD3ε:CD3γ polypeptide complex, or a CD3ε:CD3δ polypeptide complex). In some embodiments, the CD3-TCR complex association domain comprises or consists of an amino acid sequence having at least 60% (preferably ≥70%, ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%) amino acid sequence identity to SEQ ID NO: 186.

[0535] In some embodiments, the CD3-TCR complex association domain is or is derived from the CD3-TCR complex association domain of TRAC. In some embodiments, the CD3-TCR complex association domain is or is derived from the region of TRAC required for association with TCRβ, TRBC1, and / or TRBC2 (i.e., to form a TRAC:TCRβ polypeptide complex, or a TRAC:TRBC1 polypeptide complex, or a TRAC:TRBC2 polypeptide complex). In some embodiments, the CD3-TCR complex association domain comprises or consists of an amino acid sequence having at least 60% (preferably one of ≥70%, ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%) amino acid sequence identity to SEQ ID NO: 157. In some embodiments, the CD3-TCR complex association domain is derived from TRAC and further comprises a modification that promotes association with another CD3-TCR complex polypeptide. In some embodiments, the CD3-TCR complex association domain comprises or consists of an amino acid sequence having at least 60% (preferably one of ≥70%, ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99%) amino acid sequence identity to SEQ ID NO: 157 and comprises a cysteine ​​residue at the position corresponding to position 47 according to SEQ ID NO: 157. In some embodiments, the CD3-TCR complex association domain comprises or consists of an amino acid sequence having at least 60% (preferably one of ≥70%, ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) amino acid sequence identity to SEQ ID NO: 208.

[0536] In some embodiments, the CD3-TCR complex association domain is or is derived from the CD3-TCR complex association domain of TRBC1. In some embodiments, the CD3-TCR complex association domain is or is derived from a region of TRBC1 required for association with TCRα and / or TRAC (i.e., to form a TRBC1:TCRα polypeptide complex, or a TRBC1:TRAC polypeptide complex). In some embodiments, the CD3-TCR complex association domain comprises or consists of an amino acid sequence having at least 60% (preferably ≥70%, ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%) amino acid sequence identity to SEQ ID NO: 161. In some embodiments, the CD3-TCR complex association domain is derived from TRBC1 and further comprises a modification that promotes association with another CD3-TCR complex polypeptide. In some embodiments, the CD3-TCR complex association domain comprises or consists of an amino acid sequence having at least 60% (preferably one of ≥70%, ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98% or ≥99%) amino acid sequence identity to SEQ ID NO: 161 and comprises a cysteine ​​residue at the position corresponding to position 56 according to the numbering of SEQ ID NO: 161. In some embodiments, the CD3-TCR complex association domain comprises or consists of an amino acid sequence having at least 60% (preferably one of ≥70%, ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) amino acid sequence identity to SEQ ID NO: 209.

[0537] In some embodiments, the CD3-TCR complex association domain is or is derived from the CD3-TCR complex association domain of TRBC2. In some embodiments, the CD3-TCR complex association domain is or is derived from a region of TRBC2 required for association with TCRα and / or TRAC (i.e., to form a TRBC2:TCRα polypeptide complex, or a TRBC2:TRAC polypeptide complex). In some embodiments, the CD3-TCR complex association domain comprises or consists of an amino acid sequence having at least 60% (preferably ≥70%, ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%) amino acid sequence identity to SEQ ID NO: 165. In some embodiments, the CD3-TCR complex association domain is derived from TRBC2 and further comprises a modification that promotes association with another CD3-TCR complex polypeptide. In some embodiments, the CD3-TCR complex association domain comprises or consists of an amino acid sequence having at least 60% (preferably one of ≥70%, ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98% or ≥99%) amino acid sequence identity to SEQ ID NO: 165 and comprises a cysteine ​​residue at the position corresponding to position 56 according to the numbering of SEQ ID NO: 165. In some embodiments, the CD3-TCR complex association domain comprises or consists of an amino acid sequence having at least 60% (preferably one of ≥70%, ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) amino acid sequence identity to SEQ ID NO: 210.

[0538] In aspects and embodiments of the present disclosure, the recombinant CD3-TCR complex polypeptide comprises the components of the antigen binding portion as described above. This is particularly true when the recombinant CD3-TCR complex polypeptide is intended to be used together with another non-identical and complementary recombinant CD3-TCR complex polypeptide. In such aspects and embodiments, the two non-identical and complementary polypeptides preferably associate with each other to form a polypeptide complex comprising the antigen binding portion. That is, the association between the recombinant CD3-TCR complex polypeptides reconstitutes a functional antigen binding portion.

[0539] For example, in the embodiments described herein, a recombinant CD3-TCR complex polypeptide comprises a VH region of an antigen-binding portion specific for a variant Fc domain and the ECD, TMD, and ICD of TRAC (T47C) as described above, and it is contemplated that the recombinant CD3-TCR complex polypeptide is used in combination with a recombinant CD3-TCR complex polypeptide comprising a VL region of an antigen-binding portion specific for a variant Fc domain and the ECD, TMD, and ICD of TRBC1 (S56C). When expressed in a cell, the two recombinant CD3-TCR complex polypeptides associate to form a polypeptide complex comprising an Fv specific for the variant Fc domain, the Fv being formed by the VH region from the first polypeptide and the VL region from the second polypeptide.

[0540] In some aspects and embodiments of the present disclosure, a first component and a second component of an antigen binding portion as described above are provided. According to such aspects and embodiments, it will be understood that the first component and the second component of the antigen binding portion are complementary and can associate to form a (complete, functional) antigen binding portion.

[0541] In some embodiments according to the present disclosure, the component of the antigen binding portion can be or comprise a VH region of an antigen binding portion having specificity for a variant Fc domain as described above (e.g., as described herein). In some embodiments, the component of the antigen binding portion can be or comprise a VL region of an antigen binding portion having specificity for a variant Fc domain (e.g., as described herein). In preferred embodiments, the VH region and the VL region can be from the same antigen binding portion.

[0542] In some embodiments, the components of the antigen binding portion comprise or consist of VH as described above. In some embodiments, the components of the antigen binding portion comprise or consist of VL as described above. In some embodiments, the components of the antigen binding portion comprise one or more antibody heavy chain constant regions (CH). In some embodiments, the components of the antigen binding portion comprise one or more antibody light chain constant regions (CL). In some embodiments, the components of the antigen binding portion comprise CH1, CH2, and / or CH3 regions of an immunoglobulin (Ig).

[0543] In some embodiments, the CD3-TCR complex polypeptide according to the present disclosure comprises or consists of one of the following structures:

[0544] N-terminus-[Signal peptide]-[Antigen binding portion or component thereof]-[CD3-TCR complex associating domain]-C-terminus

[0545] N-terminus-[Antigen binding portion or component thereof]-[CD3-TCR complex associating domain]-C-terminus

[0546] N-terminus - [Signal peptide] - [Antigen binding portion or component thereof] - [CD3-TCR complex association domain] - [Cleavage site] - [Detectable moiety] - C-terminus

[0547] N-terminus - [Antigen binding portion or component thereof] - [CD3-TCR complex associating domain] - [Cleavage site] - [Detectable moiety] - C-terminus

[0548] In some embodiments, a composite polypeptide according to the present disclosure comprises or consists of one of the following structures:

[0549] N-terminus-[Signal peptide]-[Antigen-binding component]-[CD3-TCR complex associating domain]-[Cleavage site]-[Signal peptide]-[Antigen-binding component]-[CD3-TCR complex associating domain]-C-terminus

[0550] N-terminus-[Signal peptide]-[Antigen-binding component]-[CD3-TCR complex associating domain]-[Cleavage site]-[Signal peptide]-[Antigen-binding component]-[CD3-TCR complex associating domain]-[Cleavage site]-[Detectable moiety]-C-terminus

[0551] In some embodiments, a CD3-TCR complex polypeptide according to the present disclosure comprises or consists of (e.g., from N-terminus to C-terminus):

[0552] (1) (i) an amino acid sequence encoding a signal peptide, e.g., an amino acid sequence having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) amino acid sequence identity to SEQ ID NO: 135;

[0553] (ii) an amino acid sequence having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) amino acid sequence identity to a sequence selected from column A of Table 1; and

[0554] (iii) an amino acid sequence having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) amino acid sequence identity to a sequence selected from column B of Table 1;

[0555] The sequence selected from column A of Table 1 and the sequence selected from column B of Table 1 are selected from the same row of Table 1.

[0556] (2) (i) an amino acid sequence having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) amino acid sequence identity to a sequence selected from column A of Table 1; and

[0557] (ii) an amino acid sequence having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) amino acid sequence identity to a sequence selected from column B of Table 1;

[0558] The sequence selected from column A of Table 1 and the sequence selected from column B of Table 1 are selected from the same row of Table 1.

[0559] (3) (i) an amino acid sequence encoding a signal peptide, e.g., an amino acid sequence having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) amino acid sequence identity to SEQ ID NO: 135;

[0560] (ii) an amino acid sequence having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) amino acid sequence identity to a sequence selected from column A of Table 1;

[0561] (iii) an amino acid sequence having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) amino acid sequence identity to a sequence selected from column B of Table 1;

[0562] (iv) an amino acid sequence encoding a cleavage site, e.g., an amino acid sequence having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) amino acid sequence identity to SEQ ID NO: 141; and

[0563] (v) an amino acid sequence encoding a detectable portion, e.g., an amino acid sequence having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%) amino acid sequence identity to SEQ ID NO: 140;

[0564] The sequence selected from column A of Table 1 and the sequence selected from column B of Table 1 are selected from the same row of Table 1.

[0565] (4) (i) an amino acid sequence having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) amino acid sequence identity to a sequence selected from column A of Table 1;

[0566] (ii) an amino acid sequence having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) amino acid sequence identity to a sequence selected from column B of Table 1;

[0567] (iii) an amino acid sequence encoding a cleavage site, e.g., an amino acid sequence having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) amino acid sequence identity to SEQ ID NO: 141; and

[0568] (iv) an amino acid sequence encoding a detectable portion, e.g., an amino acid sequence having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%) amino acid sequence identity to SEQ ID NO: 140;

[0569] The sequence selected from column A of Table 1 and the sequence selected from column B of Table 1 are selected from the same row of Table 1.

[0570] Table 1

[0571]

[0572]

[0573] In some embodiments, the CD3-TCR complex polypeptide according to the present disclosure comprises or consists of an amino acid sequence having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%) amino acid sequence identity to one of SEQ ID NOs: 211 to 255. In some embodiments, the CD3-TCR complex polypeptide according to the present disclosure comprises or consists of an amino acid sequence having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%) amino acid sequence identity to SEQ ID NO: 222.

[0574] In some embodiments, a composite polypeptide according to the present disclosure comprises or consists of (e.g., from N-terminus to C-terminus):

[0575] (1) (i) an amino acid sequence encoding a signal peptide, e.g., an amino acid sequence having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) amino acid sequence identity to SEQ ID NO: 135;

[0576] (ii) an amino acid sequence having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) amino acid sequence identity to a sequence selected from column A of Table 2; and

[0577] (iii) an amino acid sequence having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) amino acid sequence identity to a sequence selected from column B of Table 2;

[0578] (iv) an amino acid sequence encoding a cleavage site, e.g., an amino acid sequence having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%) amino acid sequence identity to SEQ ID NO: 141;

[0579] (v) an amino acid sequence encoding a signal peptide, e.g., an amino acid sequence having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) amino acid sequence identity to SEQ ID NO: 135;

[0580] (vi) an amino acid sequence having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) amino acid sequence identity to a sequence selected from column C of Table 2; and

[0581] (vii) an amino acid sequence having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%) amino acid sequence identity to a sequence selected from column D of Table 2;

[0582] The sequence selected from column A of Table 2, the sequence selected from column B of Table 2, the sequence selected from column C of Table 2, and the sequence selected from column D of Table 2 are selected from the same row of Table 2.

[0583] (2) (i) an amino acid sequence encoding a signal peptide, e.g., an amino acid sequence having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) amino acid sequence identity to SEQ ID NO: 135;

[0584] (ii) an amino acid sequence having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) amino acid sequence identity to a sequence selected from column A of Table 2; and

[0585] (iii) an amino acid sequence having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) amino acid sequence identity to a sequence selected from column B of Table 2;

[0586] (iv) an amino acid sequence encoding a cleavage site, e.g., an amino acid sequence having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%) amino acid sequence identity to SEQ ID NO: 141;

[0587] (v) an amino acid sequence encoding a signal peptide, e.g., an amino acid sequence having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) amino acid sequence identity to SEQ ID NO: 135;

[0588] (vi) an amino acid sequence having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%) amino acid sequence identity to a sequence selected from column C of Table 2;

[0589] (vii) an amino acid sequence having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%) amino acid sequence identity to a sequence selected from column D of Table 2;

[0590] (viii) an amino acid sequence encoding a cleavage site, e.g., an amino acid sequence having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) amino acid sequence identity to SEQ ID NO: 141; and

[0591] (ix) an amino acid sequence encoding a detectable portion, e.g., an amino acid sequence having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%) amino acid sequence identity to SEQ ID NO: 140;

[0592] The sequence selected from column A of Table 2, the sequence selected from column B of Table 2, the sequence selected from column C of Table 2, and the sequence selected from column D of Table 2 are selected from the same row of Table 2.

[0593] Table 2

[0594]

[0595]

[0596] In some embodiments, the composite polypeptides according to the present disclosure comprise or consist of an amino acid sequence having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%) amino acid sequence identity to one of SEQ ID NOs: 256 to 279. In some embodiments, the composite polypeptides according to the present disclosure comprise or consist of an amino acid sequence having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%) amino acid sequence identity to SEQ ID NO: 260. In some embodiments, a composite polypeptide according to the present disclosure comprises or consists of an amino acid sequence having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) amino acid sequence identity to SEQ ID NO: 266.

[0597] In some embodiments, a polypeptide complex according to the present disclosure comprises a CD3-TCR complex polypeptide according to the embodiments described herein.

[0598] In some embodiments, the polypeptide complex according to the present disclosure comprises:

[0599] (a) a polypeptide comprising (e.g., from N-terminus to C-terminus):

[0600] (i) have at least 70% (preferably ≥80%),

[0601] ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%,

[0602] amino acid sequences having one of ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity; and

[0603] (ii) have at least 70% (preferably ≥80%),

[0604] ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%,

[0605] amino acid sequences with one of ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity;

[0606] as well as

[0607] (b) a polypeptide comprising (e.g., from N-terminus to C-terminus):

[0608] (i) have at least 70% (preferably ≥80%),

[0609] ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%,

[0610] amino acid sequences having one of ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity; and

[0611] (ii) have at least 70% (preferably

[0612] ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%,

[0613] amino acid sequences with one of ≥96%, ≥97%, ≥98%, ≥99%, or 100% amino acid sequence identity;

[0614] The sequence selected from column A of Table 2, the sequence selected from column B of Table 2, the sequence selected from column C of Table 2, and the sequence selected from column D of Table 2 are selected from the same row of Table 2.

[0615] In some embodiments, the polypeptide complex according to the present disclosure comprises:

[0616] (1)(i) a polypeptide comprising or consisting of an amino acid sequence having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) amino acid sequence identity to SEQ ID NO: 227; and

[0617] (ii) a polypeptide comprising, or consisting of, an amino acid sequence having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) amino acid sequence identity to SEQ ID NO: 255;

[0618] or

[0619] (2)(i) a polypeptide comprising or consisting of an amino acid sequence having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) amino acid sequence identity to SEQ ID NO: 231; and

[0620] (ii) a polypeptide comprising, or consisting of, an amino acid sequence having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) amino acid sequence identity to SEQ ID NO: 255;

[0621] or

[0622] (3)(i) a polypeptide comprising or consisting of an amino acid sequence having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) amino acid sequence identity to SEQ ID NO: 235; and

[0623] (ii) a polypeptide comprising, or consisting of, an amino acid sequence having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) amino acid sequence identity to SEQ ID NO: 255;

[0624] or

[0625] (4)(i) a polypeptide comprising or consisting of an amino acid sequence having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) amino acid sequence identity to SEQ ID NO: 239; and

[0626] (ii) a polypeptide comprising, or consisting of, an amino acid sequence having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) amino acid sequence identity to SEQ ID NO: 243;

[0627] or

[0628] (5)(i) a polypeptide comprising or consisting of an amino acid sequence having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) amino acid sequence identity to SEQ ID NO: 239; and

[0629] (ii) a polypeptide comprising, or consisting of, an amino acid sequence having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) amino acid sequence identity to SEQ ID NO: 247;

[0630] or

[0631] (6)(i) a polypeptide comprising or consisting of an amino acid sequence having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) amino acid sequence identity to SEQ ID NO: 239; and

[0632] (ii) a polypeptide comprising, or consisting of, an amino acid sequence having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) amino acid sequence identity to SEQ ID NO: 251.

[0633] In a preferred embodiment, the polypeptide complex according to the present disclosure comprises:

[0634] (i) a polypeptide comprising, or consisting of, an amino acid sequence having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) amino acid sequence identity to SEQ ID NO: 235; and

[0635] (ii) a polypeptide comprising, or consisting of, an amino acid sequence having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) amino acid sequence identity to SEQ ID NO: 255.

[0636] In a preferred embodiment, the polypeptide complex according to the present disclosure comprises:

[0637] (i) a polypeptide comprising, or consisting of, an amino acid sequence having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) amino acid sequence identity to SEQ ID NO: 239; and

[0638] (ii) a polypeptide comprising, or consisting of, an amino acid sequence having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) amino acid sequence identity to SEQ ID NO: 251.

[0639] Antigen-binding molecules (targeting antibodies) carrying variant Fc domains

[0640] In the therapeutic / prophylactic interventions of the present disclosure, molecules carrying variant Fc domains (e.g., recombinant Fc-IL2v polypeptides) are used as adaptor molecules to direct the activity of cytokines (e.g., IL2 and / or variants thereof) to cells expressing the MAB polypeptides (complexes) as described herein.

[0641] In some aspects, in the therapeutic / prophylactic interventions of the present disclosure, another molecule carrying a variant Fc domain (e.g., a targeting antibody) can be simultaneously used as an adapter molecule to direct the activity of cells expressing the MAB polypeptides (complexes) according to the present disclosure against antigens on target cells (e.g., tumor cells). That is, in embodiments where the cells are immune cells (e.g., T cells), antigen binding molecules carrying variant Fc domains can direct a cell-mediated immune response (e.g., a T cell-mediated immune response) against cells expressing the antigen to which the antigen binding molecule binds (see, e.g., Figures 8 and 9). Such antigen binding molecules carrying variant Fc domains are referred to herein as "targeting antibodies."

[0642] For example, in examples of the present disclosure, T cells expressing a CD3-TCR polypeptide complex comprising a recombinant CD3-TCR complex polypeptide according to SEQ ID NO: 222 are used together with an anti-FolR1 antibody comprising an Fc domain containing P329G, thereby directing the T cells against cells expressing FolR1. Further by way of example, in examples of the present disclosure, T cells expressing a CD3-TCR polypeptide complex comprising a recombinant CD3-TCR complex polypeptide according to SEQ ID NO: 222 are used together with an anti-CD19 antibody comprising an Fc domain containing P329G, thereby directing the T cells against cells expressing CD19. Further by way of example, in examples of the present disclosure, T cells expressing a CD3-TCR polypeptide complex comprising (i) a recombinant CD3-TCR complex polypeptide according to SEQ ID NO: 235 and (ii) a recombinant CD3-TCR complex polypeptide according to SEQ ID NO: 255 are used together with an anti-FolR1 antibody comprising an Fc domain containing P329G, thereby directing the T cells against cells expressing FolR1. By further example, in an example of the present disclosure, T cells expressing a CD3-TCR polypeptide complex comprising (i) a recombinant CD3-TCR complex polypeptide according to SEQ ID NO: 235 and (ii) a recombinant CD3-TCR complex polypeptide according to SEQ ID NO: 255 are used together with an anti-CD19 antibody comprising an Fc domain containing P329G, such that the T cells are directed against cells expressing CD19.

[0643] The targeting antibodies as described herein may comprise any of the Fc domain polypeptides as described above (eg, in an Fc domain polypeptide portion). Additionally, the targeting antibodies are capable of binding to a target antigen.

[0644] In some aspects, the targeting antibody comprises at least one antigen binding portion thereof. As used herein, "antigen binding portion thereof" refers to the part that is combined with a given target antigen. Antigen binding portion thereof includes antibodies (i.e., immunoglobulins (Ig)) and antigen binding fragments and derivatives thereof. In certain embodiments, according to the antigen binding portion thereof of the present disclosure, include monoclonal antibodies, monospecific antibodies, multispecific (e.g., bispecific, trispecific, etc.) antibodies, variable fragment (Fv) moieties, single-chain Fv (scFv) moieties, fragment antigen binding (Fab) moieties, single-chain Fab moieties (scFab), crossFab moieties, Fab' moieties, Fab'-SH moieties, F(ab')2 moieties, double antibody moieties, three antibody moieties, scFv-Fc moieties, mini antibody moieties, only heavy chain antibodies (HCAb) moieties or single domain antibodies (dAb, VHH) moieties or consist of them. Further included are target antigen binding peptides / polypeptides such as peptide aptamers, thioredoxins, anticalins, Kunitz domains, avimers, knottins, fynomers, atrimers, DARPins, affibodies, affilins, armadillo repeat proteins (ArmRPs), OBodys, and adnectins (reviewed, for example, in Reverdatto et al., Curr Top Med Chem. 2015; 15(12): 1082–1101, the entire contents of which are incorporated herein by reference (see also, for example, Boersma et al., J Biol Chem (2011) 286: 41273-85 and Emanuel et al., Mabs (2011) 3: 38-48)). Further included are target antigen binding nucleic acids, such as nucleic acid aptamers (reviewed in, for example, Zhou and Rossi Nat Rev Drug Discov. 2017 16(3):181-202). Further included are target antigen binding small molecules (e.g., low molecular weight (<1000 Daltons, typically between about 300 and 700 Daltons) organic compounds).

[0645] The antigen binding portion of the targeting antibody disclosed herein is capable of binding to the target antigen. The antigen binding portion preferably shows specific binding to the target antigen. As used herein, "specific binding" refers to selective binding to the target antigen, and it can be distinguished from non-specific binding to non-target antigens. The antigen binding portion that specifically binds to a given target antigen preferably binds to the target antigen with greater affinity and / or longer duration than when it binds to other non-target antigens. The ability of a given portion to specifically bind to a given target antigen can be determined by analyzing according to methods known in the art, such as by ELISA, surface plasmon resonance (SPR; see, for example, Hearty et al., Methods Mol Biol (2012) 907: 411-442), biolayer interferometry (BLI; see, for example, Lad et al., (2015) J Biomol Screen 20 (4): 498-507), flow cytometry, or by radiolabeled antigen binding assay (RIA) enzyme-linked immunosorbent assay. By such analysis, the binding to a given target antigen can be measured and quantified. In some embodiments, the level of binding may be the response detected in a given assay.

[0646] In some embodiments, the antigen binding moiety is expressed in the micromolar range (i.e., K D =9.9x 10 -4 to 1x 10 -6 In some embodiments, the antigen-binding portion binds to the target antigen with a submicromolar affinity (i.e., Kp) (e.g., as determined by SPR or BLI). D <1x 10 -6 In some embodiments, the antigen binding portion binds to the target antigen with an affinity in the nanomolar range (i.e., K D =9.9x 10 -7 to 1x 10 -9 In some embodiments, the antigen binding portion binds to the target antigen with subnanomolar affinity (i.e., K D <1x 10 -9 In some embodiments, the antigen binding portion binds to the target antigen with an affinity in the picomolar range (i.e., K D =9.9x 10 -10 to 1x 10 -12 In some embodiments, the antigen binding portion binds to the target antigen with sub-picomolar affinity (i.e., K D <1x 10 -12 M) binds to the target antigen.

[0647] The target antigen can be any target antigen expressed by cells that are desired to be killed / consumed to obtain a therapeutic / preventative effect. In some embodiments, the target antigen is an antigen whose expression / activity or upregulated expression / activity is positively correlated with a disease / disorder (e.g., cancer, infectious disease, or autoimmune disease). The target antigen is preferably expressed on the cell surface of cells expressing the target antigen.

[0648] In some embodiments, the target antigen can be a cancer cell antigen. A cancer cell antigen is an antigen expressed or overexpressed by a cancer cell. A cancer cell antigen can be any peptide / polypeptide, glycoprotein, lipoprotein, polysaccharide, glycolipid, lipid, or fragment thereof. The expression of a cancer cell antigen may be associated with cancer. A cancer cell antigen may be abnormally expressed by a cancer cell (e.g., a cancer cell antigen may be expressed with abnormal positioning), or may be expressed by a cancer cell with an abnormal structure. A cancer cell antigen may be able to trigger an immune response. In some embodiments, an antigen is expressed at the cell surface of a cancer cell (i.e., a cancer cell antigen is a cancer cell surface antigen). In some embodiments, a portion of an antigen bound by an antigen binding molecule as described herein is displayed on the outer surface of a cancer cell (i.e., outside the cell). A cancer cell antigen may be a cancer-associated antigen. In some embodiments, a cancer cell antigen is an antigen whose expression is related to the development, progression, or severity of a cancer symptom. A cancer-associated antigen may be associated with the etiology or pathology of a cancer, or may be abnormally expressed due to cancer. In some embodiments, a cancer cell antigen is an antigen whose expression is upregulated (e.g., at RNA and / or protein levels) by a cancer cell, for example, compared to the level expressed by a comparable non-cancerous cell (e.g., a non-cancerous cell from the same tissue / cell type). In some embodiments, cancer associated antigens may be preferentially expressed by cancer cells and not expressed by comparable non-cancerous cells (e.g., non-cancerous cells derived from the same tissue / cell type). In some embodiments, cancer associated antigens may be products of mutated oncogenes or mutated tumor suppressor genes. In some embodiments, cancer associated antigens may be products of overexpressed cellular proteins, cancer antigens produced by oncogenic viruses, carcinoembryonic antigens, or cell surface glycolipids or glycoproteins.

[0649] Cancer cell antigens are reviewed in the following literature: Zarour HM, DeLeo A, Finn OJ, et al. Categories of Tumor Antigens. In: Kufe DW, Pollock RE, Weichselbaum RR, et al., eds. Holland-Frei Cancer Medicine. 6th ed. Hamilton (ON): BC Decker; 2003. Cancer cell antigens include carcinoembryonic antigens: CEA, immature laminin receptor, TAG-72; tumor virus antigens, such as HPV E6 and E7; overexpressed proteins: BING-4, calcium-activated chloride channel 2, cyclin B1, 9D7, Ep-CAM, EphA3, HER2 / neu, telomerase, mesothelin, SAP-1, survivin; cancer testis antigens: BAGE, CAGE, GAGE, MAGE, SAGE, XAGE, CT9, CT10, NY-ESO-1, PRAME, SSX-2; lineage-restricted antigens: MART1, Gp100, tyrosinase, TRP-1 / 2, MC1R, prostate-specific antigen; mutant antigens: β-catenin, BRCA1 / 2, CDK4, CML66, fibronectin, MART-2, p53, Ras, TGF-βRII; post-translationally altered antigens: MUC1; idiotypic antigens: immunoglobulins, TCR. Other cancer cell antigens include heat shock protein 70 (HSP70), heat shock protein 90 (HSP90), glucose-regulated protein 78 (GRP78), vimentin, nucleolin, fetal pancreatic acinar protein (FAPP), alkaline phosphatase placental-like protein 2 (ALPPL-2), siglec-5, stress-induced phosphoprotein 1 (STIP1), protein tyrosine kinase 7 (PTK7) and cyclophilin B. In some embodiments, the cancer cell antigen is a cancer cell antigen described in Zhao and Cao, Front Immunol. (2019) 10: 2250, the entire contents of which are incorporated herein by reference.

[0650] In some embodiments, the target antigen is selected from the group consisting of: FAP (fibroblast activation protein), CEA (carcinoembryonic antigen), p95 (p95HER2), BCMA (B cell maturation antigen), EpCAM (epithelial cell adhesion molecule), MSLN (mesothelin), MCSP (melanoma chondroitin sulfate proteoglycan), HER-1 (human epidermal growth factor 1), HER-2 (human epidermal growth factor 2), HER-3 (human epidermal growth factor 3), CD19, CD20, CD22, CD33, CD38, CD52Flt3, folate receptor 1 (FOLR 1), human trophoblast cell surface antigen 2 (Trop-2), cancer antigen 12-5 (CA-12-5), human leukocyte antigen-antigen D related (HLA-DR), MUC-1 (mucin-1), A33 antigen, PSMA (prostate-specific membrane antigen), FMS-like tyrosine kinase 3 (FLT-3), PSMA (prostate-specific membrane antigen), PSCA (prostate stem cell antigen), transferrin-receptor, TNC (tenascin), carbonic anhydrase IX (CA-IX), and / or peptides that bind to molecules of the human major histocompatibility complex (MHC). In some embodiments, the target antigen is CD19. In some embodiments, the target antigen is FOLR1.

[0651] It will be understood that the cells and compositions of the present disclosure can be used in the treatment / prevention of any disease / disorder that would derive therapeutic or prophylactic benefit from a reduction in the level / activity of a given target antigen, or a reduction in the number / proportion / activity of cells containing / expressing a given target antigen.

[0652] For example, the disease / disorder can be a disease / disorder in which the target antigen or cells comprising / expressing the target antigen are pathologically involved, such as a disease / disorder in which an increase in the level / activity of the target antigen, or an increase in the number / ratio / activity of cells comprising / expressing the target antigen, is positively correlated with the onset, development or progression of the disease / disorder and / or the severity of one or more symptoms of the disease / disorder. In some embodiments, an increase in the level / activity of the target antigen, or an increase in the number / ratio / activity of cells comprising / expressing the target antigen, can be a risk factor for the onset, development or progression of the disease / disorder.

[0653] In some embodiments, the disease / disorder to be treated / prevented according to the present disclosure is characterized by an increase in the expression or activity level of the target antigen, for example, compared to the expression / activity level in the absence of the disease / disorder. In some embodiments, the disease / disorder to be treated / prevented is a disease / disorder characterized by an increase in the number / ratio / activity of cells expressing the target antigen, for example, compared to the level / number / ratio / activity in the absence of the disease / disorder (e.g., in a healthy subject or in an equivalent non-diseased tissue). When the disease / disorder is cancer, the level of expression or activity of the target antigen may be higher than the level of expression or activity of the target antigen in an equivalent non-cancerous cell / non-tumor tissue. Cancer / its cells may comprise one or more mutations (e.g., relative to an equivalent non-cancerous cell / non-tumor tissue) that cause an increase in the expression or activity of the target antigen.

[0654] Therapeutic / prophylactic intervention according to the present disclosure can achieve one or more of the following in a subject (compared to an equivalent untreated subject or a subject treated with an appropriate control): a reduction in the level of the target antigen; a reduction in the activity of the target antigen; and / or a reduction in the number / proportion / activity of cells containing / expressing the target antigen.

[0655] Uses of cells and compositions

[0656] In particular, the cells and compositions according to the present disclosure are contemplated for use in methods of treating / preventing diseases / disorders by adoptive cell transfer (ACT).

[0657] Adoptive cell transfer generally refers to the process of obtaining cells (such as immune cells) from a subject, usually by extracting a blood sample from the isolated cells.Then, the cell is usually modified and / or amplified, and then applied to the same subject (in the case of adoptive transfer of autologous / autologous cells) or applied to different subjects (in the case of adoptive transfer of allogeneic cells). Treatment is usually intended to provide a cell mass with some desired characteristics to the subject, or increase the frequency of such cells with such characteristics in the subject. Adoptive transfer can be carried out for the purpose of introducing cells or cell mass into the subject and / or increasing the frequency of cells or cell mass in the subject.

[0658] Adoptive transfer of immune cells is described, for example, in Kalos and June (2013), Immunity 39(1):49-60 and Davis et al. (2015), Cancer J. 21(6):486–491, both of which are incorporated herein by reference in their entirety. A skilled artisan will be able to determine appropriate reagents and procedures for adoptive transfer of cells based on this disclosure, for example, with reference to Dai et al., 2016 J Nat Cancer Inst 108(7):djv439, the entirety of which is incorporated by reference.

[0659] The cells and compositions according to the present disclosure can be used to treat / prevent diseases / disorders by allogeneic transplantation or autologous transplantation.

[0660] As used herein, "allografting" refers to the transplantation of cells, tissues or organs that are genetically different from the recipient subject to the recipient subject. The cells, tissues or organs may come from or may be derived from cells, tissues or organs of a donor subject that is genetically different from the recipient subject. Allogeneic transplantation is different from autologous transplantation, and autologous transplantation refers to the transplantation of cells, tissues or organs (i.e., autologous material) from / derived from a donor subject that is genetically identical to the recipient subject. It should be understood that the adoptive transfer of allogeneic immune cells is a form of allogeneic transplantation, and the adoptive transfer of autologous immune cells is a form of autologous transplantation.

[0661] The present disclosure provides methods comprising administering to a subject the cells and compositions according to the present disclosure.

[0662] In some embodiments, the methods comprise modifying an immune cell to comprise / express a polypeptide according to the disclosure (eg, a recombinant MAB polypeptide).

[0663] In some embodiments, the methods comprise modifying an immune cell to express or comprise a MAB polypeptide according to the disclosure (eg, as described herein), and administering the modified immune cell to a subject.

[0664] In some embodiments, the method further comprises administering to the subject (i) a recombinant Fc domain-IL2 variant (Fc-IL2v) polypeptide complex comprising a variant Fc domain according to the disclosure, and / or (ii) a targeting antibody comprising a variant Fc domain according to the disclosure, wherein the recombinant MAB polypeptide comprises an antigen binding portion that binds to the recombinant Fc-IL2v polypeptide complex and / or the variant Fc domain of the targeting antibody.

[0665] It will be appreciated that the method steps described in the preceding three paragraphs may be performed in any suitable order.

[0666] In some embodiments, the method comprises administering to a subject an immune cell modified to express or comprise a recombinant MAB polypeptide, wherein the recombinant MAB polypeptide is a chimeric antigen receptor (CAR) according to the present disclosure.

[0667] In some embodiments, the methods comprise administering to a subject immune cells modified to express or comprise a recombinant MAB polypeptide comprising one or more recombinant CD3-TCR complex polypeptides according to the disclosure.

[0668] In some embodiments, according to the preceding two paragraphs, the subject is a subject who (i) has been administered or is to be administered a recombinant Fc domain-IL2 variant (Fc-IL2v) polypeptide complex comprising a variant Fc domain according to the disclosure and / or (ii) a targeting antibody comprising a variant Fc domain according to the disclosure, wherein the recombinant MAB polypeptide comprises an antigen binding portion that binds to the variant Fc domain.

[0669] In some embodiments, the method comprises:

[0670] (a) modifying an immune cell to express or comprise a recombinant MAB polypeptide according to the disclosure (e.g., as described herein); and

[0671] (b) administering to the subject (i) a recombinant Fc domain-IL2 variant (Fc-IL2v) polypeptide complex comprising a variant Fc domain according to the disclosure and / or (ii) a targeting antibody comprising a variant Fc domain according to the disclosure; and

[0672] (c) administering the modified immune cells to a subject;

[0673] wherein the recombinant MAB polypeptide of (a) comprises an antigen binding portion bound to the variant Fc domain of (b).

[0674] In some embodiments of the method according to the preceding paragraph, step (c) may be performed before step (b).

[0675] In some embodiments, the subject from which the immune cells are isolated / obtained is the same subject to which the cells are administered (i.e., the adoptive transfer can be autologous / autologous cells). In some embodiments, the subject from which the immune cells are isolated / obtained is a different subject from the subject to which the cells are administered (i.e., the adoptive transfer can be allogeneic cells).

[0676] In some embodiments, the method may further include one or more of the following:

[0677] (i) obtaining a blood sample from a subject;

[0678] (ii) isolating immune cells (e.g., PBMCs) from a blood sample that has been obtained from a subject;

[0679] (iii) generating / expanding immune cell populations;

[0680] (iv) culturing immune cells in vitro or ex vivo cell culture;

[0681] (v) culturing immune cells expressing / comprising the recombinant MAB polypeptides according to the disclosure in vitro or ex vivo cell culture;

[0682] (vi) collecting / isolating immune cells expressing / comprising the recombinant MAB polypeptides according to the present disclosure;

[0683] (vii) formulating the immune cells expressing / comprising the recombinant MAB polypeptides according to the disclosure into a pharmaceutical composition, for example, by mixing the cells with a pharmaceutically acceptable adjuvant, diluent or carrier.

[0684] The administration of the articles of the present disclosure is preferably carried out in a "therapeutically effective" or "preventively effective" amount, which is sufficient to show therapeutic or preventive benefits to the subject. The actual amount applied, as well as the rate and time course of administration, will depend on the nature and severity of the disease / disorder and the specific article being administered. Prescribing treatment, such as determining the dosage, is the responsibility of general practitioners and other doctors, and generally takes into account the disease / disorder to be treated, the condition of the individual subject, treatment site delivery, method of administration, and other factors known to practitioners. Examples of the above techniques and protocols can be found in Remington's The Science and Practice of Pharmacy (ed. A. Adejare), 23rd edition (2020), Academic Press.

[0685] The administration of the articles of the present disclosure can be parenteral, systemic, intravenous, intraarterial, intramuscular, intracavitary, intrathecal, intraocular, intravitreal, intraconjunctival, subretinal, suprachoroidal, subcutaneous, intradermal, intrathecal, oral, nasal, topical or transdermal. Administration can be carried out by injection or infusion. Administration of the articles of the present disclosure can be intratumoral. In some cases, the articles of the present disclosure can be formulated for targeted delivery to specific cells, tissues, organs and / or tumors.

[0686] Multiple doses of the articles of the present disclosure can be provided. The multiple doses can be separated by a predetermined time interval, which can be selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days, or one of 1, 2, 3, 4, 5, or 6 months.

[0687] Administration of cells or compositions according to the present disclosure to a subject according to the therapeutic and prophylactic interventions described herein can be simultaneous or sequential.

[0688] Simultaneous administration refers to administering (i) a cell or composition according to the present disclosure and (ii) an antigen binding molecule described herein together, e.g., as a pharmaceutical composition containing both agents (i.e., a combined preparation), or immediately sequentially, and optionally via the same route of administration, e.g., into the same artery, vein, or other blood vessel.

[0689] Sequential administration refers to the administration of one of (i) a cell or composition according to the present disclosure and (ii) an antigen binding molecule as described herein, followed by separate administration of the other agent after a given time interval. It is not necessary to administer both agents by the same route, although in some embodiments the same route is used. The time interval can be any time interval.

[0690] The present disclosure also provides a method for depleting or killing cells comprising or expressing a target antigen, the method comprising contacting the cells comprising / expressing the target antigen with:

[0691] (i) an antigen binding molecule comprising: (a) an antigen binding domain that binds to a target antigen, and (b) a variant Fc domain according to the present disclosure; and

[0692] (ii) an immune cell comprising / expressing a recombinant MAB polypeptide according to the disclosure;

[0693] wherein the MAB polypeptide of (ii) comprises an antigen binding portion that binds to the variant Fc domain of the antigen binding molecule of (i).

[0694] Nucleic acids and vectors

[0695] The present disclosure provides a nucleic acid or multiple nucleic acids encoding a recombinant MAB polypeptide, a recombinant Fc-IL2v polypeptide complex, or a targeting antibody according to the present disclosure. In some embodiments, the nucleic acid comprises or consists of DNA and / or RNA.

[0696] The MAB polypeptides, recombinant Fc-IL2v polypeptide complexes, or targeting antibodies according to the present disclosure can be produced in a cell by translating RNA encoding the recombinant MAB polypeptides, recombinant Fc-IL2v polypeptide complexes, or targeting antibodies. The recombinant MAB polypeptides, recombinant Fc-IL2v polypeptide complexes, or targeting antibodies according to the present disclosure can be produced in a cell by transcription of nucleic acids encoding the recombinant MAB polypeptides, recombinant Fc-IL2v polypeptide complexes, or targeting antibodies and subsequent translation of the transcribed RNA.

[0697] In some embodiments, the nucleic acid can be a vector or vectors, or can be contained in a vector or vectors.As used herein, a "vector" is a nucleic acid molecule that serves as a vehicle for transferring exogenous nucleic acid into a cell.

[0698] Thus, the present disclosure also provides a vector or vectors comprising a nucleic acid or nucleic acids according to the present disclosure. The vector can facilitate delivery of a nucleic acid encoding a recombinant MAB polypeptide, a recombinant Fc-IL2v polypeptide complex, or a targeting antibody according to the present disclosure to a cell. The vector can be an expression vector comprising the elements required for expression of a recombinant MAB polypeptide, a recombinant Fc-IL2v polypeptide complex, or a targeting antibody according to the present disclosure. The vector can comprise elements that facilitate integration of the nucleic acid into the genomic DNA of a cell into which the vector is introduced.

[0699] Nucleic acids and vectors according to the present disclosure can be provided in purified or isolated form, ie, from other nucleic acids or naturally occurring biological material.

[0700] The vector can be a vector (i.e., an expression vector) for expressing nucleic acids in cells. Such vectors can include a promoter sequence operably linked to a nucleotide sequence encoding a recombinant MAB polypeptide, a recombinant Fc-IL2v polypeptide complex, or a targeting antibody. The vector can also include a stop codon (i.e., located 3' of the nucleotide sequence encoding the recombinant MAB polypeptide, the recombinant Fc-IL2v polypeptide complex, or the targeting antibody in the nucleotide sequence of the vector) and an expression enhancer. Any suitable vector, promoter, enhancer, and stop codon known in the art can be used to express a peptide or polypeptide from a vector according to the present disclosure.

[0701] The term "operably linked" can include situations where a nucleic acid encoding a recombinant MAB polypeptide, a recombinant Fc-IL2v polypeptide complex, or a targeting antibody according to the present disclosure and a regulatory nucleic acid sequence (e.g., a promoter and / or enhancer) are covalently linked in such a manner that expression of the nucleic acid encoding the recombinant MAB polypeptide, a recombinant Fc-IL2v polypeptide complex, or a targeting antibody is placed under the influence or control of the regulatory nucleic acid sequence (thereby forming an expression cassette). Thus, a regulatory sequence is operably linked to a selected nucleic acid sequence if it is capable of influencing the transcription of the nucleic acid sequence. The resulting transcript can then be translated into the desired polypeptide.

[0702] Vectors contemplated for use in connection with the present disclosure include DNA vectors, RNA vectors, plasmids (e.g., conjugative plasmids (e.g., F plasmids), non-conjugative plasmids, R plasmids, col plasmids, episomes), viral vectors (e.g., retroviral vectors, e.g., gammaretroviral vectors (e.g., murine leukemia virus (MLV)-derived vectors, e.g., SFG vectors), lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, vaccinia viral vectors, and herpes viral vectors), transposon-based vectors, and artificial chromosomes (e.g., yeast artificial chromosomes), as described, for example, in Maus et al., Annu Rev Immunol (2014) 32: 189-225 and Morgan and Boyerinas, Biomedicines (2016) 4: 9, the entire contents of which are incorporated herein by reference. In some embodiments, the vector according to the present disclosure is a lentiviral vector.

[0703] In some embodiments, the vector can be a eukaryotic vector, i.e., a vector comprising the elements necessary for expressing a protein from the vector in a eukaryotic cell. In some embodiments, the vector can be a mammalian vector, e.g., comprising a cytomegalovirus (CMV) or SV40 promoter to drive protein expression.

[0704] In some embodiments, the nucleic acid / nucleic acids or vector / vectors according to the present disclosure comprise an EF1α promoter.

[0705] In some embodiments, the nucleic acid / nucleic acids or vector / vectors according to the present disclosure encode a CAR comprising or consisting of an amino acid sequence having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) amino acid sequence identity to one of SEQ ID NO: 146, SEQ ID NO: 149, SEQ ID NO: 151 and SEQ ID NO: 154. In some embodiments, the nucleic acid / nucleic acids or vector / vectors according to the present disclosure encode a CAR comprising or consisting of an amino acid sequence having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) amino acid sequence identity to SEQ ID NO: 152.

[0706] In a preferred embodiment, the nucleic acid / nucleic acids or vector / vectors according to the present disclosure comprise the nucleotide sequence of SEQ ID NO: 152, or a codon-degenerate nucleotide sequence thereof encoding the amino acid sequence encoded by SEQ ID NO: 152.

[0707] In some embodiments, the nucleic acid / nucleic acids or vector / vectors according to the present disclosure encode a CD3-TCR complex polypeptide comprising or consisting of an amino acid sequence having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) amino acid sequence identity to one of SEQ ID NOs: 211 to 255. In some embodiments, the nucleic acid / nucleic acids or vector / vectors according to the present disclosure encode a CD3-TCR complex polypeptide comprising or consisting of an amino acid sequence having at least 70% (preferably one of ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%) amino acid sequence identity to SEQ ID NO: 222.

[0708] In a preferred embodiment, the nucleic acid / nucleic acids or vector / vectors according to the present disclosure comprise the nucleotide sequence of SEQ ID NO: 287, or a codon-degenerate nucleotide sequence thereof encoding the amino acid sequence encoded by SEQ ID NO: 287.

[0709] As used herein, a "codon-degenerate nucleotide sequence" of a reference nucleotide sequence refers to a nucleotide sequence that has nucleotides that are not identical to those of the reference nucleotide sequence, but that, as a result of the degeneracy of the genetic code, encodes the same amino acid sequence as that encoded by the reference nucleotide sequence.

[0710] The constituent polypeptides of the polypeptide complex according to the present disclosure may be encoded by different nucleic acids among the multiple nucleic acids according to the present disclosure, or encoded by different vectors of the multiple nucleic acids according to the present disclosure.

[0711] In aspects and embodiments of the present disclosure, one or more nucleic acids according to the present disclosure encode two or more (e.g., 2, 3, 4 or more) recombinant CD3-TCR complex polypeptides according to the present disclosure. In aspects and embodiments of the present disclosure, one or more vectors according to the present disclosure encode two or more (e.g., 2, 3, 4 or more) recombinant CD3-TCR complex polypeptides according to the present disclosure.

[0712] In some embodiments where the nucleic acid / nucleic acids or vector / vectors encode two or more (e.g., 2, 3, 4, or more) recombinant CD3-TCR complex polypeptides, the recombinant CD3-TCR complex polypeptides are not identical. In some embodiments, the nucleic acid / nucleic acids or vector / vectors encode complementary recombinant CD3-TCR complex polypeptides. That is, in some embodiments, the nucleic a...

Claims

1. A recombinant Fc domain-IL2 variant (Fc-IL2v) polypeptide complex in combination with a recombinant membrane-anchored antigen binding (MAB) polypeptide or MAB polypeptide complex for use in treating cancer, preventing or treating metastasis, or stimulating an immune response or function such as T cell activity. The recombinant Fc-IL2v polypeptide complex comprises: (i) a first polypeptide comprising a variant CH2-CH3 region comprising G329 according to EU numbering, and wherein the first polypeptide further comprises an IL-2 variant (IL2v) polypeptide comprising an IL-2 polypeptide comprising amino acid substitutions F42A, Y45A, and L72G, wherein the numbering is relative to the human IL-2 sequence SEQ ID NO: 40; and (ii) a second polypeptide comprising a variant CH2-CH3 region comprising G329 according to EU numbering, wherein the recombinant Fc-IL2v polypeptide complex does not comprise an antigen binding portion, and wherein the MAB polypeptide or MAB polypeptide complex comprises an antigen binding portion or a component thereof, and a transmembrane domain, wherein the antigen binding portion binds to a variant CH2-CH3 region comprising an amino acid substitution P329G according to EU numbering, relative to the amino acid sequence of a reference CH2-CH3 region comprising P329 according to EU numbering, to which the antigen binding portion does not bind.

2. A recombinant Fc domain-IL2 variant (Fc-IL2v) polypeptide complex comprising: (i) a first polypeptide comprising a variant CH2-CH3 region comprising G329 according to EU numbering, and wherein the first polypeptide further comprises an IL-2 variant (IL2v) polypeptide comprising an IL-2 polypeptide comprising amino acid substitutions F42A, Y45A, and L72G, wherein the numbering is relative to the human IL-2 sequence SEQ ID NO: 40; and (ii) a second polypeptide comprising a variant CH2-CH3 region comprising G329 according to EU numbering, wherein the recombinant Fc-IL2v polypeptide complex does not comprise an antigen binding portion.

3. A method for treating or preventing cancer in an individual or for stimulating an immune response or function such as T cell activity in an individual, wherein the method comprises (a) administering to the individual a recombinant Fc domain-IL2 variant (Fc-IL2v) polypeptide complex, wherein the recombinant Fc-IL2v polypeptide complex comprises: (i) a first polypeptide comprising a variant CH2-CH3 region comprising G329 according to EU numbering, and wherein the first polypeptide further comprises an IL-2 variant (IL2v) polypeptide comprising an IL-2 polypeptide comprising amino acid substitutions F42A, Y45A, and L72G, wherein the numbering is relative to the human IL-2 sequence SEQ ID NO: 40; and (ii) a second polypeptide comprising a variant CH2-CH3 region comprising G329 according to EU numbering; and (b) administering a recombinant membrane-anchored antigen binding (MAB) polypeptide or MAB polypeptide complex, wherein the MAB polypeptide or MAB polypeptide complex comprises an antigen binding portion, or a component thereof, and a transmembrane domain, wherein the antigen binding portion binds to a variant CH2-CH3 region comprising an amino acid substitution P329G according to EU numbering, relative to the amino acid sequence of a reference CH2-CH3 region comprising P329 according to EU numbering, to which the antigen binding portion does not bind.

4. Use of a recombinant Fc domain-IL2 variant (Fc-IL2v) polypeptide complex in the manufacture of a medicament for treating or preventing cancer in an individual, or for stimulating an immune response or function, such as T cell activity, in an individual, wherein the recombinant Fc-IL2v polypeptide complex comprises: (i) a first polypeptide comprising a variant CH2-CH3 region comprising G329 according to EU numbering, and wherein the first polypeptide further comprises an IL-2 variant (IL2v) polypeptide comprising an IL-2 polypeptide comprising amino acid substitutions F42A, Y45A, and L72G, wherein the numbering is relative to the human IL-2 sequence SEQ ID NO: 40; and (ii) a second polypeptide comprising a variant CH2-CH3 region comprising G329 according to EU numbering.

5. Use of a recombinant Fc domain-IL2 variant (Fc-IL2v) polypeptide complex in the manufacture of a medicament for treating or preventing cancer in an individual, or for stimulating an immune response or function, such as T cell activity, in an individual, wherein the treatment comprises: (a) administering a recombinant Fc domain-IL2 variant (Fc-IL2v) polypeptide complex to the individual, The recombinant Fc-IL2v polypeptide complex comprises: (i) a first polypeptide comprising a variant CH2-CH3 region comprising G329 according to EU numbering, and wherein the first polypeptide further comprises an IL-2 variant (IL2v) polypeptide comprising an IL-2 polypeptide comprising amino acid substitutions F42A, Y45A, and L72G, wherein the numbering is relative to the human IL-2 sequence SEQ ID NO: 40; and (ii) a second polypeptide comprising a variant CH2-CH3 region comprising G329 according to EU numbering; and (b) administering a recombinant membrane-anchored antigen binding (MAB) polypeptide or MAB polypeptide complex, wherein the MAB polypeptide or MAB polypeptide complex comprises an antigen binding portion, or a component thereof, and a transmembrane domain, wherein the antigen binding portion binds to a variant CH2-CH3 region comprising an amino acid substitution P329G according to EU numbering, relative to the amino acid sequence of a reference CH2-CH3 region comprising P329 according to EU numbering, to which the antigen binding portion does not bind.

6. The recombinant Fc-IL2v polypeptide complex, method or use of any preceding claim, wherein the antigen binding portion that binds to Fc-IL2v comprises the heavy chain variable (VH) region and the light chain variable (VL) region of an antibody that binds to the variant CH2-CH3 region comprising the amino acid substitution P329G according to EU numbering.

7. The recombinant Fc-IL2v polypeptide complex, method or use according to any one of the preceding claims, wherein the antigen binding portion is or comprises an Fv, scFv, Fab, Fab', Fab'-SH, F(ab')2, crossFab, scFab or dAb portion.

8. The recombinant Fc-IL2v polypeptide complex, method or use according to any one of the preceding claims, wherein the antigen binding portion comprises: (a)(i) a VH region comprising the following CDRs: an HC-CDR1 having the amino acid sequence of SEQ ID NO: 11; an HC-CDR2 having the amino acid sequence of SEQ ID NO: 19; and an HC-CDR3 having the amino acid sequence of SEQ ID NO: 13; as well as (ii) a VL region comprising the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 24; LC-CDR2 having the amino acid sequence of SEQ ID NO: 25; and an LC-CDR3 having the amino acid sequence of SEQ ID NO: 26; or (b)(i) a VH region comprising the following CDRs: an HC-CDR1 having the amino acid sequence of SEQ ID NO: 11; an HC-CDR2 having the amino acid sequence of SEQ ID NO: 12; and an HC-CDR3 having the amino acid sequence of SEQ ID NO: 13; as well as (ii) a VL region comprising the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 24; LC-CDR2 having the amino acid sequence of SEQ ID NO: 25; and LC-CDR3 having the amino acid sequence of SEQ ID NO:

26.

9. The recombinant Fc-IL2v polypeptide complex, method or use according to any one of the preceding claims, wherein the mutant IL-2 polypeptide further comprises the amino acid substitution Q126T.

10. The recombinant Fc-IL2v polypeptide complex, method or use according to any one of the preceding claims, wherein the recombinant Fc-IL2v polypeptide complex does not comprise an antigen binding portion, in particular wherein the recombinant Fc-IL2v polypeptide complex does not comprise scFv, Fab or crossFab.

11. The recombinant Fc-IL2v polypeptide complex, method or use of any preceding claim, wherein the recombinant MAB polypeptide comprises an amino acid sequence derived from IL2Ra, IL15Ra or CD8a.

12. The recombinant Fc-IL2v polypeptide complex, method or use according to any one of the preceding claims, wherein the recombinant MAP polypeptide is a chimeric antigen receptor (CAR).

13. The recombinant Fc-IL2v polypeptide complex, method or use according to any one of the preceding claims, wherein the recombinant MAP polypeptide comprises at least one recombinant CD3-TCR complex polypeptide.

14. The recombinant Fc-IL2v polypeptide complex, method or use according to any one of the preceding claims 13, wherein the recombinant CD3-TCR complex polypeptide comprises: (i) an antigen binding portion or component thereof, wherein the antigen binding portion binds to a variant CH2-CH3 region comprising an amino acid substitution, P329G, according to EU numbering, relative to the amino acid sequence of a reference CH2-CH3 region comprising P329 according to EU numbering, to which the antigen binding portion does not bind; and (ii) a CD3-TCR complex association domain having an amino acid sequence derived from a CD3-TCR complex polypeptide.

15. The recombinant Fc-IL2v polypeptide complex, method or use according to claim 14, wherein the recombinant CD3-TCR complex polypeptide is capable of associating with one or more CD3-TCR complex polypeptides via its CD3-TCR complex association domain to form a CD3-TCR complex.

16. The recombinant Fc-IL2v polypeptide complex, method or use according to claim 14 or 15, wherein the amino acid sequence derived from the CD3-TCR complex polypeptide is derived from CD3ε, TCRα or TCRβ.

17. A cell comprising the recombinant MAB polypeptide or MAB polypeptide complex according to any one of claims 1 or 11 to 16.

18. A method for producing an enriched cell pool, the method comprising: A starting cell pool comprising at least one cell according to claim 17 is contacted with the recombinant Fc-IL2v polypeptide complex according to any one of claims 2 or 6 to 10, and the cells are incubated until the proportion of cells comprising the recombinant MAB polypeptide or MAB polypeptide complex reaches the desired proportion of the total cell pool to produce the enriched cell pool.

19. A nucleic acid or nucleic acids encoding a recombinant Fc domain-IL2 variant (Fc-IL2v) polypeptide complex according to any one of claims 2 or 6 to 10 or a recombinant MAB polypeptide or MAB polypeptide complex according to any one of claims 1 or 11 to 16.

20. An expression vector or a plurality of expression vectors comprising the nucleic acid according to claim 19.

21. A cell comprising: a recombinant MAB polypeptide or MAB polypeptide complex according to any one of claims 1 or 11 to 16; a nucleic acid or nucleic acids according to claim 19; or an expression vector or expression vectors according to claim 20.

22. The recombinant Fc-IL2v polypeptide complex, method or use according to any one of claims 1 to 16, or the cell according to claim 21, wherein cells expressing the recombinant MAB polypeptide and / or recombinant MAB polypeptide complex cells are specifically expanded, in particular wherein the cells are specifically expanded by contacting the cells with the recombinant Fc-IL2v polypeptide complex according to any one of claims 2 or 6 to 10.

23. The recombinant Fc-IL2v polypeptide complex, method or use according to any one of claims 1 to 16, or the cell according to claim 21, wherein cells expressing the recombinant MAB polypeptide and / or recombinant MAB polypeptide complex cells are enriched, in particular wherein the cells are enriched by contacting the cells with the recombinant Fc-IL2v polypeptide complex according to any one of claims 2 or 6 to 10.

24. The recombinant Fc-IL2v polypeptide complex, method or use according to any one of claims 1 to 16, or the cell according to claim 21, wherein cells expressing the recombinant MAB polypeptide and / or recombinant MAB polypeptide complex cells are enriched to >90% of the total cell pool.

25. A method for producing an enriched cell pool, the method comprising: A starting cell pool comprising at least one cell according to claim 21 is contacted with the recombinant Fc-IL2v polypeptide complex according to any one of claims 2 or 6 to 10, and the cells are incubated until the proportion of cells comprising the recombinant MAB polypeptide or MAB polypeptide complex reaches the desired proportion of the total cell pool to produce the enriched cell pool.

26. A pharmaceutical composition comprising: the recombinant Fc domain-IL2 variant (Fc-IL2v) polypeptide complex according to any one of claims 2 or 6 to 10; the cell according to claim 21; or the enriched cell pool produced according to claim 25.

27. The invention as hereinbefore described with reference to the accompanying drawings and examples.

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