Immune-activating Fc domain binding molecule

Abstract

The present invention generally relates to novel immunostimulatory Fc domain-binding molecules for activating and redirecting immune cells to specific target cells. The present invention further relates to polynucleotides encoding such molecules, as well as vectors and host cells comprising such polynucleotides. The present invention further relates to methods for producing the bispecific antigen-binding molecules of the present invention and methods for using these bispecific antigen-binding molecules in the treatment of disease.

Description

[Technical Field]

[0001] Field of Invention This invention generally relates to novel immunoactivating Fc domain binding molecules for the activation of immune cells and redirection to specific target cells. Furthermore, this invention relates to polynucleotides encoding such molecules, as well as vectors and host cells containing such polynucleotides. This invention further relates to methods for producing the bispecific antigen-binding molecules of the present invention, and methods for using these bispecific antigen-binding molecules in the treatment of diseases. [Background technology]

[0002] background Selective destruction of individual cells or specific cell types is often desirable in a variety of clinical situations. For example, the primary goal of cancer treatment is to specifically destroy tumor cells while leaving healthy cells and tissues intact and undamaged, or to destroy specific cell subsets identified by specific surface antigens.

[0003] An attractive way to achieve this is to induce an immune response against target cells by recruiting immune effector cells such as natural killer (NK) cells, monocytes / macrophages, or cytotoxic T lymphocytes (CTLs) to attack and destroy tumor cells.

[0004] One method for inducing immune effector cell-mediated target cell death or depletion is antibody-dependent cellular cytotoxicity (ADCC) or antibody-dependent cellular cytotoxicity (ADCP) mediated by IgG1 isotype ADCC-competent antibodies and antibodies with enhanced ADCC effector function (Zahavi et al, AntibodyTherapeutics, 1, 7-12 (2018)). Alternatively, T cells can be recruited to kill target cells via bispecific T cell antibodies designed to bind to surface antigens on target cells and have a second binding site to the invariant component that activates the T cell receptor (TCR) complex (Clynes and Desjarlais, Annu Rev Med 70:427-450 (2019)). These include BiTE (bispecific T cell engagers) (Nagorsen and Baeuerle, Exp Cell Res 317, 1255-1260 (2011)), diabodies (Holliger et al., Prot Eng 9, 299-305 (1996)), DART (biaffinity retargeting) (Moore et al., Blood 117, 4542-51 (2011)), or so-called 2+1 T cell bispecific antibodies (TCBs) (Bacac et al., Clin Cancer Res Several bispecificity formats, including 24, 4785-4797 (2018), have been developed, and their suitability for T cell-mediated immunotherapy is being investigated.

[0005] The various formats under development demonstrate significant potential stemming from the redirection and activation of immune cells in immunotherapy. To date, developed bispecific antibodies consistently engage directly with the desired antigen, thereby linking target cells and CTLs and resulting in target cell lysis. These bispecific antibody formats face challenges related to toxicity, applicability, and productivity. Furthermore, for each single target (combination), it is necessary to construct individual molecules specific to each target. The therapeutic utility of antibodies and their derivatives is not limited to their function as T-cell engagers; indications are also found in the modulation of checkpoints of inhibitors or activators. For example, the use of immune checkpoint inhibitor antibodies has demonstrated persistent responses in several indications (Hodie et al. N Engl J Med.;363(8):711-23.(2010); Prieto PA, et al. Clin cancer Res.;18:2039-2047(2012)). More recently, it has been shown that the activity of T cell bispecific antibodies can be further enhanced by bispecific substances that activate so-called costimulatory pathways on T cells via the activation of CD28 (Skokos et al., Sci Trans Med 12(525):1-14(2020)) or 4-1BB signaling (Claus et al., Sci Trans Med 11(496), eaav5989(2019)).

[0006] In addition to current successes, therapies are limited in their flexibility to target multiple different antigens and their ability to selectively utilize combined NK or CTL functions in a single application. [Overview of the project]

[0007] Summary of the Invention An immunoactivating fragment crystallizable (Fc) domain binding molecule, (a) an Fc domain binding moiety that specifically binds to a target Fc domain containing at least one amino acid substitution of the first set, (b) Immune activation portion and Immunoactivating Fc domain-binding molecules, including the above, are provided herein.

[0008] In one embodiment, at least one amino acid substitution in the first set reduces binding to the Fc receptor and / or reduces effector function.

[0009] In one embodiment, the immune-activating Fc domain binding molecule is (c) Further containing a half-life extension Fc domain, The Fc domain binding portion does not specifically bind to the half-life extension Fc domain.

[0010] In one embodiment, the half-life extension Fc domain contains at least one amino acid of a second set. In one embodiment, the substitution of at least one amino acid of the second set reduces binding to Fc. In one embodiment, the target Fc domain and / or the half-life extension Fc domain is composed of first and second subunits capable of stable association.

[0011] In one embodiment, the target Fc domain and / or half-life extension Fc domain is an IgG1 or IgG4Fc domain.

[0012] In one embodiment, the target Fc domain exhibits reduced binding affinity to the Fc receptor and / or reduced effector function compared to the native IgG1Fc domain.

[0013] In one embodiment, the half-life extended Fc domain exhibits reduced binding affinity to the Fc receptor and / or reduced effector function compared to the natural IgG1Fc domain.

[0014] In one embodiment, at least one amino acid substitution in the first set reduces the binding affinity and / or effector function to the Fc receptor, and at least one amino acid substitution in the second set includes one or more amino acid substitutions at the same amino acid position as at least one amino acid substitution in the first set, and the amino acids in at least one amino acid substitution in the second set are substituted with different amino acids at the same position compared to at least one amino acid substitution in the first set.

[0015] In one embodiment, at least one amino acid substitution in the second set reduces the binding affinity to the Fc receptor and / or effector function.

[0016] In one embodiment, at least one amino acid substitution in the first set includes at least one amino acid substitution at a position selected from the list consisting of 233, 234, 235, 238, 253, 265, 269, 270, 297, 310, 331, 327, 329 and 435 (numbering according to Kabat's EU index).

[0017] In one embodiment, at least one amino acid substitution in the second set includes at least one amino acid substitution at a position selected from the list consisting of 233, 234, 235, 238, 253, 265, 269, 270, 297, 310, 331, 327, 329 and 435 (numbering according to Kabat's EU index).

[0018] In one embodiment, at least one amino acid substitution in the first set includes the amino acid substitution P329G (numbered according to the Kabat EU index), and at least one amino acid substitution in the second set includes a substitution at position P329 (numbered according to the Kabat EU index) by an amino acid other than glycine (G).

[0019] In one embodiment, at least one amino acid substitution in the second set includes a substitution at position P329 (numbered according to the Kabat EU index) by an amino acid selected from the list consisting of arginine (R), leucine (L), isoleucine (I), and alanine (A).

[0020] In one embodiment, at least one amino acid substitution of the second set includes a substitution by arginine (R) at position P329 (numbered according to Kabat's EU index). [Brief explanation of the drawing]

[0021] [Figure 1]An example illustrating the concept of the present invention: A targeted antibody containing at least one antigen-binding moiety capable of specific binding to target cells is combined with an immunoactivating Fc domain-binding molecule to generate a versatile set of ready-made molecules for human therapeutics. The targeted antibody contains at least one amino acid substitution in its Fc domain (hereinafter referred to as the targeted Fc domain), and the immunoactivating Fc domain-binding molecule is capable of specific binding to an Fc domain containing such an amino acid substitution (hereinafter referred to as the first set of at least one amino acid substitution). The immunoactivating Fc domain-binding molecule is capable of specific binding to the targeted antibody (containing at least one amino acid substitution of the first set) via the antigen-binding moiety, which is hereafter referred to herein as the Fc domain-binding moiety. The immunoactivating Fc domain-binding molecule further comprises an immunoactivating moiety (e.g., an antigen-binding moiety capable of specific binding to CD3, CD28, or 4-1BB) and / or, for example, a cytokine (e.g., IL2) and / or a co-stimulatory ligand (e.g., 4-1BBL). An immune-activating Fc domain binding molecule can activate immune cells (e.g., T cells) via this immune-activating moiety. The immune-activating Fc domain binding molecule may also contain an Fc domain, which will hereafter be referred to as a half-life extension Fc domain (to distinguish it from a target Fc domain). A half-life extension Fc domain may also contain at least one amino acid substitution (e.g., reducing effector function), which will hereafter be referred to as a second set of at least one amino acid substitution (to distinguish it from a first set of at least one amino acid substitution). To avoid binding of the immune-activating Fc domain binding molecule to other (identical) immune-activating Fc domain binding molecules, the Fc domain binding moiety does not have the ability to specifically bind to a half-life extension Fc domain. In this concept, antibody therapies that invoke immune cells can be specifically adapted with respect to the desired effector function during treatment and / or over time. Figure 43 shows a diagram of an exemplary therapeutic toolbox provided below. [Figure 2]These are exemplary structures of the (multispecific) antibody of the present invention. (A, D) Diagram of a "1+1 CrossMab" molecule. (B, E) Diagram of a "2+1 IgG Crossfab" molecule with the order of the Crossfab and Fab components reversed ("reversed"). (C, F) Diagram of a "2+1 IgG Crossfab" molecule. (G, K) Diagram of a "1+1 IgG Crossfab" molecule with the order of the Crossfab and Fab components reversed ("reversed"). (H, L) Diagram of a "1+1 IgG Crossfab" molecule. (I, M) Diagram of a "2+1 IgG Crossfab" molecule having two CrossFabs. (J, N) Diagram of a "2+1 IgG Crossfab" molecule having two CrossFabs with the order of the Crossfab and Fab components reversed ("reversed"). (O, S) Diagram of a "Fab-Crossfab" molecule. (P, T) Diagram of a "Crossfab-Fab" molecule. (Q, U) Diagram of the "(Fab)2-Crossfab" molecule. (R, V) Diagram of the "Crossfab-(Fab)2" molecule. (W, Y) Diagram of the "Fab-(Crossfab)2" molecule. (X, Z) Diagram of the "(Crossfab)2-Fab" molecule. ++, --: Amino acids of opposite charge optionally introduced in the CH1 and CL domains. The Crossfab molecule is shown as including the exchange of the VH and VL regions, but in embodiments where no charge modification is introduced in the CH1 and CL domains, it alternatively includes the exchange of the CH1 and CL domains. [Figure 3]Binding of huIgG1 P329x variant to captured recombinant human Fcg receptor. (A) Setup; Recombinant FcgRs are captured by an anti-His antibody immobilized on the chip surface. In the second step, huIgG1 P329x variants at concentrations of 150, 300 and 600 nM are injected and their interaction with FcgRs is analyzed. (B) Sensorgram showing binding of huIgG1 P329x variant to huFcgRIa. (C) Sensorgram showing binding of huIgG1 P329x variant to huFcgRIIa. (d) Sensorgram showing binding of huIgG1 P329x variant to huFcgRIIb. (E) Sensorgram showing binding of huIgG1 P329x variant to huFcgRIIIa. [Figure 4] (A) Assay setup for the binding of huIgG1 P329x LALA variant to anti-P329G antibody; anti-P329G (M-1.7.24) antibody was bound to the surface of the sensor chip. In the second step, the huIgG1 P329x variant was injected at a concentration of 500 nM (performed in triplicate). HuIgG1 P329G was used as a positive control. (B) Sensorgram showing the interaction of huIgG1 P329L with anti-P329G (M-1.7.24) antibody (triplicate). (C) Sensorgram showing the interaction of huIgG1 P329I with anti-P329G (M-1.7.24) antibody (triplicate). (D) Sensorgram showing the interaction of huIgG1 P329R with anti-P329G (M-1.7.24) antibody (triplicate). (E) Sensorgram showing the interaction of huIgG1 P329A with anti-P329G (M-1.7.24) antibody (performed in a triple sequence). [Figure 5](A) Schematic diagram of the T cell bispecific antibody (TCB) molecule used in the example. All TCB antibodies tested were prepared as "2+1 IgG CrossFab, inverted type" with charge modification (VH / VL exchange in CD3 binder, charge modification in target cell antigen binder, EE=147E,213E;RK=123R,124K). (B~E) Components for TCB assembly: light chain of anti-TYRP1 Fab molecule with charge modification at CH1 and CL (B), light chain of anti-CD3 crossover Fab molecule (C), heavy chain with knob and PG LALA mutation in the Fc region (D), heavy chain with hole and PG LALA mutation in the Fc region (E). [Figure 6] Schematic diagram of the surface plasmon resonance (SPR) setup used in Example 3. Anti-PG antibody bound to the C1 sensor chip. Human and cynomolgus monkey CD3 (fused to the Fc region) are passed over the surface to analyze the interaction between the anti-CD3 antibody and CD3 in the TCB. [Figure 7] TCBs containing optimized anti-CD3 antibody were tested in a Jurkat NFAT reporter assay using CHO-K1 TYRP1 clone 76 as the target cell. They were compared with TCBs containing CD3orig. Activation of Jurkat NFAT reporter cells was determined by measuring luminescence 4 hours (A) and 24 hours (B) after treatment. [Figure 8] Tumor cell killing of melanoma cell line M150543, derived from healthy donor PBMCs, was evaluated when treated with a TCB containing either an optimized anti-CD3 antibody or the parental binder CD3orig. Tumor cell killing was measured by quantitative analysis of LDH release at 24 hours (A) and 48 hours (B). [Figure 9] Upregulation of CD25 and CD69 on CD8 T cells (A,B) and CD4 T cells (C,D) was analyzed in healthy donor-derived PBMCs treated with either an optimized anti-CD3 antibody or a TCB containing parental binder CD3orig, in the presence of the M150543 melanoma cell line as the target cell line. Analysis was performed by flow cytometry after 48 hours. [Figure 10]CD25 expression on CD8 (A) and CD4 T cells (B) was analyzed in healthy donor-derived PBMCs treated with either an optimized anti-CD3 antibody or a TCB containing the parental binder CD3orig, in the absence of tumor target cells. Analysis was performed by flow cytometry after 48 hours. [Figure 11] (A) Schematic diagram of the monovalent IgG molecule produced in Example 19. The monovalent IgG molecule was produced as human IgG1 by VH / VL exchange in a CD3 binder. (B-E) Components for the assembly of monovalent IgG: light chain of anti-CD3 crossover Fab molecule (B), heavy chain with a knob and PG LALA mutation in the Fc region (C), and heavy chain with a hole and PG LALA mutation in the Fc region (D). [Figure 12] (A) Exemplary configuration of the T cell activating bispecific antigen-binding molecule (TCB) of the present invention. Exemplary anti-P329G xCD3 1+1 universal TCB (uTCB). (B) Exemplary configuration of the binding mode of the 1+1 uTCB to P329G mutations of tumors targeting IgG and T cell receptor (TCR) on T cells. ++, --: amino acids with opposite charges introduced in the CH and CL domains. [Figure 13] (A) Exemplary configuration of the T cell activating bispecific antigen-binding molecule (TCB) of the present invention. Exemplary anti-P329G xCD3 2+1 universal TCB (uTCB). (B) Exemplary configuration of the binding mode of the 2+1 uTCB to P329G mutations in tumors targeting IgG and T cell receptor (TCR) on T cells. The 2+1 uTCB format can simultaneously bind to two tumor-targeting antibodies having P32G mutations. ++, --: amino acids with opposite charges introduced in the CH and CL domains. [Figure 14]Figure 14 shows schematic diagrams of different immunoactivating Fc-binding molecules having an anti-CD3 effector moiety (other effector moieties can be used in the same format, i.e., anti-CD3 effector moieties, e.g., anti-CD28, anti-4-1BB can be replaced). The half-life extended Fc domain contains the P329x mutation, where x is an amino acid other than glycine (G). 14A: 1+1 format, anti-P329G, cross-anti-CD3, charged variant KK / EE, P329x, LALA, knob / hole. 14B: Classical 2+1 format, anti-P329G, cross-anti-CD3, charged, P329x, LALA, knob / hole. 14C / D: Inverted 2+1 format, anti-P329G, cross-anti-CD3, charged, P329x, LALA, knob / hole. [Figure 15] A) Anti-P329G(VH3VL1)xCD3(CH2527)1+1 TCB can simultaneously bind to immobilized human CD3 epsilon-delta-Fc and huFc(P329G); B) Anti-P329G(VH3VL1)xCD3(P035.093)1+1 TCB can simultaneously bind to immobilized human CD3 epsilon-delta-Fc and huFc(P329G); C) Anti-P329G(VH3VL1)xCD3(P035.093)2+1 TCB can simultaneously bind to immobilized human CD3 epsilon-delta-Fc and huFc(P329G). Three injections. [Figure 16] Kinetic activation of T cells by various concentrations of anti-P329G(M-1.7.24)xCD3(CH2527)2+1 TCBs combined with various concentrations of anti-FolR1(6D5)P329G LALA huIgG1 antibody. Evaluation was performed by quantifying the intensity of CD3 downstream signaling using a Jurkat-NFAT reporter assay. Three series technical mean values ​​are shown. Error bars indicate standard deviation (SD). [Figure 17]Kinetic activation of T cells by various concentrations of anti-P329G(M-1.7.24)xCD3(CH2527)2+1 TCBs combined with various concentrations of anti-CD20(GA101)P329G LALA huIgG1 antibody. Evaluation was performed by quantifying the intensity of CD3 downstream signaling using a Jurkat-NFAT reporter assay. Three series technical mean values ​​are shown, and error bars indicate standard deviation (SD). [Figure 18] Kinetic activation of T cells by various concentrations of anti-P329G(M-1.7.24)xCD3(CH2527)2+1 TCBs combined with various concentrations of anti-FAP(4B9)P329G LALA huIgG1 antibody. Evaluation was performed by quantifying the intensity of CD3 downstream signaling using a Jurkat-NFAT reporter assay. Three series technical mean values ​​are shown, and error bars indicate standard deviation (SD). [Figure 19] T cell activation by various concentrations of anti-P329G(M-1.7.24)xCD3(CH2527)2+1 TCBs combined with anti-CD20(GA101)P329G LALA huIgG1 antibody. Either CD20+z-138 (Figure 6A) or CD20+SU-DHL-4 cells were used as target cells. CD3 downstream signaling intensity was evaluated by quantification using a Jurkat-NFAT reporter assay. Three series technical mean values ​​are shown, and error bars indicate SD. [Figure 20] Specific dose-dependent activation of T cells in the presence of a tumor-targeted anti-CD20 (GA101) antibody carrying a P329G mutation, combined with anti-P329G(M-1.7.24)xCD3(CH2527)2+1 TCB. Anti-CD20 wild-type huIgG1 or anti-CD20 LALA mutant huIgG1 did not activate T cells. This was evaluated by quantifying the intensity of CD3 downstream signaling using a Jurkat-NFAT reporter assay. Three consecutive technical mean values ​​are shown, and error bars indicate SD. [Figure 21]Reduction in the number of target cells in adherent tumor cells in the presence of anti-P329G(M-1.7.24)xCD3(CH2527)2+1 TCBs combined with tumor-targeted anti-EpCAM (Figure 21A), anti-STEAP (Figure 21B), or anti-FAP (4B9) (Figure 21C). Evaluation was performed by quantitative analysis of red nuclear cell count over time. Three series of technical mean values ​​are shown, and error bars indicate SD. [Figure 22] T cell activation by different uTCB formats. 1+1 uTCB or 2+1 uTCB containing mouse or humanized P329G binder and different CD3 binders. Evaluation was performed by quantifying the intensity of CD3 downstream signaling using a Jurkat-NFAT reporter assay. Either FolR1+ HeLa cells (Figure 22A) or CD19+ SU-DHL-4 cells (Figure 22B) were used as target cells. Triple-sequence technical mean values ​​are shown, and error bars indicate SD. [Figure 23] FolR1+HeLa target cell lysis (E:T ratio 5:1) was performed using human PBMCs and uTCB in 1+1 uTCB or 2+1 uTCB with a humanized P329G binder. The ratio of uTCB to P329G LALA IgG1 was 1:2. Tumor cell lysis was evaluated at 5.5 hours, 20 hours, and 42 hours by calorimetry of lactate dehydrogenase (LDH) release. The technical mean values ​​of the three series are shown. Error bars indicate standard deviation (SD). [Figure 24] CD19+Nalm6 target cells were lysed using human PBMCs and uTCB (E:T ratio 5:1) in 1+1 uTCB or 2+1 uTCB with humanized P329G binder and CD3 binder P035.093. The ratio of uTCB to P329G LALA IgG1 was 1:2. Tumor cell lysis was evaluated at 5.5 hours, 20 hours, and 42 hours by calorimetry of lactate dehydrogenase (LDH) release. The technical mean values ​​of the three series are shown, and error bars indicate standard deviation (SD). [Figure 25] Examples of immune-activating Fc-binding molecules containing anti-PG and anti-CD28 moieties. [Figure 26]The immobilized anti-P329G(M-1.7.24)xCD28(TGN1412_var 15_crossover)1+1 can simultaneously bind to human IgG(P329G) and human CD28-Fc. Dual injection. [Figure 27] Binding analysis of a bispecific antigen-binding molecule to human CD28 overexpressed on CHO transfectant cells. The relative median (MFI) and standard deviation (SD) of three fluorescence values ​​are shown. The EC50 value of binding was calculated using GraphPadPrism. [Figure 28] IL2 reporter cell assay after 4-hour incubation, determined by luminescence. 25,000 IL2-reporter effector cells were incubated with fixed-concentration 625 pM CD3 IgG (PGLALA-containing Fc) in or without increasing concentrations of PG-CD28 (8.4 pM to 34.4 nM). As a control, PG-CD28 was included in the presence of an isotype control (including PGLALA-containing Fc), and each tumor-targeted CD28 molecule was not crosslinked in this assay setting due to the absence of a tumor target. Relative luminescence (RLU) was determined as a direct measurement of Jurkat activation after 4 hours. RLU values ​​from triplicates are shown along with standard deviation (SD). [Figure 29] (A) An immunoactivating Fc-binding molecule with an IL2v (cytokine) effector moiety, and (B) a schematic diagram showing that anti-P329G(M-1.7.24)xIL2v hugG1 can simultaneously bind to immobilized huIL2R-Fc and huFc(P329). Three injections. (C) IL-2 signaling (STAT5-P) shown as the frequency of STAT5-P in human PD1+CD4 T cells after 12 minutes of exposure to an IL-2v-based molecule. Mean ± SEM of two donors. (D) IL-2 signaling (STAT5-P) shown as MFI of STAT5-P in human PD1+CD4 T cells after 12 minutes of exposure to an IL-2v-based molecule. Mean ± SEM of two donors. [Figure 30]The components for constructing monovalent P329G targeted the split trimer human 4-1BB ligand. A) A dimer ligand fused to the human IgG1-CL domain. B) A monomeric ligand fused to the human IgG1-CH1 domain. [Figure 31] A monovalent P329G-targeting split trimer 4-1BB ligand Fc(kih)LALA fusion containing a CH-CL crossover with a charged residue, also known as anti-P329Gx4-1BBL huIgG1. *Charged residue [Figure 32] a) Setting of the simultaneous binding of anti-P329G(M-1.7.24)x4-1BBL huIgG1 to hu4-1BB and huIgG1-P329G; b) Simultaneous binding of anti-P329G(M-1.7.24)x4-1 BBL huIgG1 to hu4-1BB-Fc(kih) and human IgG1 containing the P329G mutation in Fc. Double replicate tests are shown. [Figure 33] B-cell depleted PBMCs were incubated with WSU DLCL2 for 3 days in the presence of grofitamab (CD20-TCB, 1nM), anti-P329Gx4-1BBL (1nM), or a combination of both. Tumor cell lysis was determined by LDH release (left) and T cell activation by flow cytometry (right, e.g., CD4+ T cells, day 3, median fluorescence intensity). [Figure 34] The bispecific antigen-binding molecule is in the huIgG1 LALA format, containing two anti-4-1BB Fab fragments (divalently bound to 4-1BB) and one anti-P329G cross-Fab fragment (a Fab fragment in which the VH and VL regions are swapped), fused at the C-terminus of the heavy chain of one of the 4-1BB Fab fragments to the N-terminus. This format is referred to herein as the 2+1 format. Large black dots represent knob-to-hole mutations, while small black dots within the CH1 / CL domain represent amino acid mutations that improve the correct pairing of the heavy chain with the anti-4-1BB light chain. [Figure 35]Different assay settings were compared with each other. The anti-P329G(M-1.7.24)x4-1BBL huIgG1 molecule was tested for its functionality using a Jurkat reporter cell line assay. Therefore, tumor target (Her2, CEACAM5, FAP) expressing cells (KPL4, MKN45, NIH / 3T3-huFAP clone 19) were co-incubated for 5 hours with human 4-1BB receptor expressing Jurkat reporter cells (Jurkat-hu4-1BB-NFkB-luc2) and various concentrations of tumor target (TT)-specific human IgG1 P329G LALA antibody, either in the presence or absence of anti-P329G(M-1.7.24)x4-1BBL huIgG1. Subsequently, luciferase activity was measured by adding a detection solution (One-Glo) and measuring the luminescence released during luciferase-mediated oxidation (Figure 5A). This activity was directly compared with that of directly tumor-targeted TT-4x1BBL huIgG1 (Figure 5B) as a positive control. [Figure 36] Testing of various ratios between anti-P329G(M-1.7.24)x4-1BBL huIgG1 and tumor target-specific huIgG1 P329G LALA. The anti-P329G(M-1.7.24)x4-1BBL huIgG1 molecule was tested for its functionality using a Jurkat reporter cell line assay, thereby maintaining the molecule in solution or crosslinking it by adding Her2+KPL4 human breast cancer cells (Figure 6A). Direct tumor target Her2x4-1BBL huIgG1 was compared with indirectly crosslinked anti-P329G(M-1.7.24)x4-1BBL huIgG1. As a result, anti-Her2 huIgG1 P329G LALA functioned as a linker between the tumor target Her2 and anti-P329G(M-1.7.24)x4-1BBL huIgG1, thereby maintaining a stable ratio of anti-Her2 huIgG1 P329G LALA to anti-P329G(M-1.7.24)x4-1BBL huIgG1 (Figure 6A). The same setup was tested with CEACAM5+MKN45 gastric cancer cells and a CEACAM5-specific antibody (Figure 6B). [Figure 37]The anti-P329G(M-1.7.24)x4-1BBL huIgG1 molecule was tested for its functionality using a Jurkat reporter cell line assay, thereby determining whether the molecule remained in solution or was crosslinked by the addition of Her2+KPL4 human breast cancer cells (Figure 7A). Direct tumor-targeted Her2x4-1BBL huIgG1 was compared to indirectly crosslinked anti-P329G(M-1.7.24)x4-1BBL huIgG1 linked by anti-Her2-specific huIgG1 P329G LALA given in a 1:2 ratio, and remained stable. Further unbound (DP47) molecules were included as controls. The same was repeated using CEACAM5+MKN45 gastric cancer cells (Figure 7B) and FAP+NIH / 3T3-huFAP clone 19 fibroblasts (Figure 7C). [Figure 38] (A) Exemplary examples of ADCC-competent IgG1 effector molecules capable of binding to P329G mutations (anti-P329G IgG1) of tumor targeting molecules (e.g., IgG1, SM). (B) Exemplary configurations of binding modes of anti-P329G IgG1 effector molecules to tumor P329G mutations targeting IgG and FcγIII on immune effector cells. [Figure 39] Antibody-dependent cell-mediated cytotoxicity (ADCC) mediated by anti-P329G(VH3VL1)huIgG1 with glycosylated Fc(GE) in the presence of tumors targeting IgG1 with the P329G LALA mutation. This was evaluated by quantitative lactate dehydrogenase (LDH) release from target cells. Technical triplicate means are shown, and error bars indicate SD. For statistical analysis, a one-way ANOVA analysis with Bonferroni correction was performed. As p-values, the New England Journal of Medicine style was used, as listed in GraphPadPrism 7. *=P≦0.033;**=P≦0.002;***=P≦0.001. [Figure 40]Downregulation of CD16 receptors on NK cells activated by anti-P329G(VH3VL1)huIgG1 in combination with tumors targeting IgG1 with the P329G LALA mutation. Evaluated by flow cytometry. Mean values ​​from technical triplicates are shown, and error bars indicate SD. For statistical analysis, a one-way ANOVA analysis with Bonferroni correction was performed. As p-values, the New England Journal of Medicine style was used, as listed in GraphPadPrism 7. *=P≦0.033;**=P≦0.002;***=P≦0.001. [Figure 41] Upregulation of CD107 on NK cells activated by anti-P329G(VH3VL1)huIgG1 in combination with tumors targeting IgG1 with the P329G LALA mutation. Evaluated by flow cytometry. Mean values ​​from technical triplicates are shown, and error bars indicate SD. For statistical analysis, a one-way ANOVA analysis with Bonferroni correction was performed. As p-values, the New England Journal of Medicine style was used, as listed in GraphPadPrism 7. *=P≦0.033;**=P≦0.002;***=P≦0.001. [Figure 42]Only the combination of anti-FAP(clone 4B9) human IgG1 P329GLALA and anti-P329G human IgG1 mAb induces dose-dependent NFAT activation (a measure of ADCC ability) in Jurkat FcγRIIIa reporter cells. Each point represents the mean of technical replication in one experiment. The standard error of the mean is shown in the error bars. (A) Fixed concentration (10 μg / mL) anti-FAP(4B9)P329G LALA huIgG1 was used in combination with an 8-fold tapering titration of anti-P329G huIgG1 mAb. (B) An 8-fold tapering titration of anti-FAP(4B9)P329G LALA huIgG1 was used in combination with a fixed concentration (10 μg / mL) anti-P329G huIgG1. Anti-P329G huIgG1 was tested as fully fucosylated (triangular) and defucosylated (circular) human IgG1 isotypes. [Figure 43] The following is an example of an exemplary therapeutic toolbox provided: A targeted antibody capable of specific binding to target cells (for therapeutic use) is combined with different immunoactivating Fc domain binding moieties capable of specific binding to the P329G mutation within the Fc domain of the targeted antibody. Provided effector functions include glycosylated Fc domains (e.g., ADCC), anti-CD3 (e.g., T cell activation), 4-1BBL and / or anti-4-1BB (e.g., T cell costimulation), anti-CD28 (e.g., T cell costimulation), and IL2v (e.g., T cell proliferation). The effector functions can be combined and / or titrated over time to optimal concentrations to maximize therapeutic benefits. [Figure 44] An example configuration for cis-targeting of PD-1 positive T cells. A targeted antibody containing the P329G mutation, capable of specific binding to PD1, is combined with an immunoactivating Fc domain-binding molecule containing the IL2v immunoactivating moiety. [Figure 45]Kinetic activation of T cells by various concentrations of anti-FOLR1 P329G LALA huIgG1 and anti-P329G(VH3VL1)xCD3(P035.093)2+1 TCB, P329R LALA Fc (molar ratio IgG:TCB 2:1). TCB concentrations used: 0nM (Figure 45A), 0.05nM (Figure 45B), 5nM (Figure 45C). HeLa(FOLR1+) cells were used as target cells. Evaluation was performed by quantifying the intensity of CD3 downstream signaling using a Jurkat-NFAT reporter assay. The technical mean values ​​of three sequences are shown. Error bars indicate SD. [Figure 46] T cell activation by anti-FOLR1 P329G LALA huIgG1 and anti-P329G(VH3VL1)xCD3(P035.093)2+1 TCB LALA Fc (molar ratio IgG:TCB 2:1) against several FOLR1+ target cell lines. HeLa (Figure 46A), JAR (Figure 46B), OVCAR-3 (Figure 46C), and SKOV-3 (Figure 46D) were used as target cells. HeLa(FOLR1+) cells were used as target cells. CD3 downstream signaling intensity was evaluated by quantification using a Jurkat-NFAT reporter assay. The technical mean values ​​of three series are shown. Error bars indicate SD. [Figure 47] T cell activation by anti-FOLR1 P329G LALA huIgG1 and anti-P329G(VH3VL1)xCD3(P035.093)2+1 TCB (molar ratio IgG:TCB 2:1) containing LALA Fc or P329R LALA Fc. HeLa(FOLR1+) cells were used as target cells. CD3 downstream signaling intensity was evaluated by quantification using a Jurkat-NFAT reporter assay. The technical mean values ​​of three sequences are shown. Error bars indicate standard deviation. [Figure 48]Primary human T cell activation, measured by CD25 upregulation on CD8+ T cells, was measured in the presence of anti-P329G(VH3VL1)xCD3(P035.093)2+1 TCB and anti-FOLR1 P329G LALA huIgG1 containing either LALA Fc or P329R LALA Fc (molar ratio IgG:TCB 2:1). Either pan T cells (Figure 48A, C) or PBMCs (Figure 48B, D) from a healthy donor were used as effector cells. Either SKOV-3(FOLR1+) (Figure 48A, B) or no target cells (Figure 48C, D) were used. Analysis was performed by flow cytometry after 48 hours. The technical mean values ​​of three consecutive sets are shown. Error bars indicate standard deviation (SD). [Figure 49] T cell activation by tumor-targeted P329G LALA huIgG1 containing anti-P329G(VH3VL1)xCD3 2+1 TCB P329R LALA Fc (molar ratio IgG:TCB 2:1) along with P035.093, CH2527, or clone 22 as a CD3 binder. This was performed against several targets and several target cells. The following were used as target cell pairs: CD19+SU-DHL-8 cells (Figure 49A), FOLR1+HeLa cells (Figure 49B), CEA+MKN-45 cells (Figure 49C), HER2+LNCaP cells (Figure 49D), and STEAP1+LNCaP cells (Figure 49E). The intensity of CD3 downstream signaling was evaluated by quantification using a Jurkat-NFAT reporter assay. The technical mean values ​​of three series are shown. Error bars indicate SD. [Figure 50] Dynamics of tumor cell lysis by primary human pan T cells in the presence of anti-FOLR1 (Figure 50A) or anti-CEA (Figure 50B) P329G LALA huIgG1 and anti-P329G(VH3VL1)×CD3(P035.093)2+1TCBP329R LALA Fc. HeLa NLR(FOLR1+)(Figure 50A) and MKN-45 NLR(Figure 50B) were used as target cells. Evaluation was performed by quantitative measurement of red nuclear cell count over time. The technical mean values ​​of three series are shown. Error bars indicate standard deviation (SD). [Figure 51]The dynamics of tumor cell lysis by primary human pan T cells in the presence of anti-FOLR1 P329G LALA huIgG1 and anti-P329G(VH3VL1)xCD3 2+1 TCB P329R LALA Fc (molar ratio IgG:TCB 2:1), along with CD3 binders P035.093, CH2527, or clone 22. HeLa NLR(FOLR1+) was used as the target cell. Evaluation was performed by quantitative measurement of red nuclear cell count over time. The technical mean values ​​of three series are shown. Error bars indicate SD. [Figure 52] Primary human T cell activation was measured by CD69 upregulation on CD8+ T cells in the presence of anti-FOLR1 P329G LALA huIgG1 and anti-P329G(VH3VL1)xCD3 2+1 TCB P329R LALA Fc (molar ratio IgG:TCB 2:1), along with P035.093, CH2527, or clone 22 as CD3 binders. Pan T cells from three healthy donors (Donor A (Figure 52A), Donor B (Figure 52B), Donor C (Figure 52C)) were used as effector cells. HeLa(FOLR1+) was used as the target cell. Analysis was performed by flow cytometry after 48 hours. The technical mean of three consecutive values ​​is shown. Error bars indicate SD. [Figure 53] Activation of 4-1BB reporter T cells by the co-stimulatory molecules anti-P329G(VH3VL1)x4-1BBBL LALA huIgG1,1+1 and anti-P329G(VH3VL1)xCD3(P035.093)2+1 TCB P329R LALA Fc in the presence of 100 nM anti-CEA P329G LALA huIgG1 and 0.5 nM anti-P329G(VH3VL1)x4-1BBBL LALA huIgG1,1+1 and anti-P329G(VH3VL1)xCD28 LALA huIgG1,1+1. SKOV-3 huCEA(CEA+) cells were used as target cells. The intensity of 4-1BB downstream signaling was evaluated by quantification using the Jurkat-NFκB reporter assay. 3 series technical mean values ​​are shown. Error bars indicate SD. [Modes for carrying out the invention]

[0022] Detailed description of the invention definition Terms are used herein in the manner they are commonly used in the art unless otherwise defined below.

[0023] As used herein, the term “antigen-binding molecule” refers in its broadest sense to a molecule that specifically binds to an antigenic determinant. Examples of antigen-binding molecules are immunoglobulins and their derivatives (e.g., fragments).

[0024] For the purposes of this specification, “acceptor human framework” is a framework comprising the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human consensus framework, as defined below. An acceptor human framework “derived” from a human immunoglobulin framework or a human consensus framework may contain the same amino acid sequence or may contain a modification of the amino acid sequence. In some embodiments, the number of amino acid modifications is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In some embodiments, the VL acceptor human framework is sequence-identical to the VL human immunoglobulin framework sequence or the human consensus framework sequence.

[0025] The term "bispecific" means that an antigen-binding molecule can specifically bind to at least two distinct antigenic determinants. Typically, a bispecific antigen-binding molecule contains two antigen-binding sites, each specific to a different antigenic determinant. In certain embodiments, a bispecific antigen-binding molecule can bind simultaneously to two antigenic determinants (in particular, two antigenic determinants expressed in two distinct cells).

[0026] As used herein, “T cell activating antigen” refers to an antigenic determinant expressed on the surface of T lymphocytes, particularly cytotoxic T lymphocytes, that can induce T cell activation through interaction with an antigen-binding molecule. Specifically, the interaction of an antigen-binding molecule with a T cell activating antigen can induce T cell activation by triggering a cascade of signaling in the T cell receptor complex. In certain embodiments, the activating T cell antigen is CD3, particularly the epsilon subunit of CD3 (see UniProt number P07766 (version 130), NCBI RefSeq number NP_000724.1; or UniProt number Q95LI5 (version 49), NCBI GenBank number BAB 71849.1).

[0027] "Affinity" refers to the strength of the total non-covalent interaction between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects the 1:1 interaction between the members of a binding pair (e.g., an antibody and an antigen). The affinity of molecule X for its partner Y is generally expressed by the dissociation constant (K). D ) can be expressed by. Affinity can be measured by methods common in the art, including those described herein. Specific illustrative and exemplary methods for measuring binding affinity are described below.

[0028] An "affinity-matured" antibody is an antibody that, compared to an unchanged parent antibody, exhibits one or more changes in one or more complementarity-determining regions (CDRs), thereby improving the antibody's affinity for an antigen.

[0029] As used herein, the term “amino acid mutation” encompasses amino acid substitutions, deletions, insertions, and modifications. Any combination of substitutions, deletions, insertions, and modifications can be performed to reach the final construct, insofar as the final construct has the desired characteristics, e.g., decreased binding to the Fc receptor or increased association with another peptide. Deletions and insertions of amino acid sequences include deletions and insertions of amino acids at the amino-terminus and / or carboxy-terminus. A specific amino acid mutation is an amino acid substitution. For example, non-conservative amino acid substitutions, i.e., replacing one amino acid with another amino acid with different structural and / or chemical properties, are particularly preferred for the purpose of altering the binding properties of the Fc region. Amino acid substitutions include substitutions with non-natural amino acids or substitutions with naturally occurring amino acid derivatives of 20 standard amino acids (e.g., 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine, 5-hydroxylysine). Amino acid mutations can be generated using genetic or chemical methods well known in the art. Genetic methods may include mutagenesis of specific sites, PCR, gene synthesis, etc. Methods for altering the side chain groups of amino acids by means other than genetic engineering, such as chemical modification, may also be useful. Various names may be used herein to describe the same amino acid mutation.

[0030] The term "antibody" is used herein in its broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, as long as they exhibit the desired antigen-binding activity.

[0031] An "antibody fragment" refers to a molecule other than an intact antibody, which contains a portion of an intact antibody that binds to an antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv and scFab); single-domain antibodies (dAb); and multispecific antibodies formed from antibody fragments. For a review of specific antibody fragments, see Holliger and Hudson, Nature Biotechnology 23:1126-1136 (2005).

[0032] The term "antigen-binding domain" refers to a portion of an antibody that specifically binds to some or all of a particular antigen and is complementary to some or all of that antigen. The antigen-binding domain may be provided, for example, by one or more antibody-variable domains (also called antibody-variable regions). In particular, the antigen-binding domain includes an antibody light chain variable domain (VL) and an antibody heavy chain variable domain (VH).

[0033] An "antigen-binding site" refers to the part of an antigen-binding molecule that interacts with an antigen, i.e., one or more amino acid residues. For example, the antigen-binding site of an antibody contains amino acid residues from the complementarity-determining region (CDR). Natural immunoglobulin molecules typically contain two antigen-binding sites, while Fab molecules typically have one antigen-binding site.

[0034] As used herein, the term “antigen-binding moiety” refers to a polypeptide molecule that specifically binds to an antigenic determinant. In one embodiment, an antigen-binding moiety can direct the entity to which it binds (e.g., a second antigen-binding moiety) to a target site, such as a specific type of tumor cell or tumor stroma having an antigenic determinant. In another embodiment, an antigen-binding moiety can activate signaling through its target antigen, such as a T cell receptor complex antigen. An antigen-binding moiety includes an antibody and its fragments as further defined herein. A particular antigen-binding moiety includes an antigen-binding domain of an antibody, comprising an antibody heavy chain variable region and an antibody light chain variable region. In a particular embodiment, an antigen-binding moiety may include an antibody constant region as further defined herein and known in the art. Useful heavy chain constant regions include any of five isotypes: α, δ, ε, γ, or μ. Useful light chain constant regions include any of two isotypes: κ and λ.

[0035] As used herein, the term “antigenic determinant” is synonymous with “antigen” and “epitope,” and refers to a site on a polypeptide macromolecule to which an antigen-binding moiety binds, forming an antigen-antigen complex (e.g., a contiguous extension of amino acids or a conformational structure composed of discontinuous amino acids from different regions). Useful antigenic determinants may be found, for example, on the surface of tumor cells, on the surface of virus-infected cells, on the surface of other diseased cells, on the surface of immune cells, free in serum, and / or in the extracellular matrix (ECM). Unless otherwise specified, the protein referred to as an antigen herein may be any native form of protein from any vertebrate source, including mammals, e.g., primates (e.g., humans) and rodents (e.g., mice and rats). In certain embodiments, the antigen is a human protein. When referring to a particular protein of the present invention, the term encompasses “full-length” untreated proteins and any form of protein obtained from cell treatment. The term also encompasses naturally occurring protein variants, e.g., splice variants or allele variants.

[0036] Antibody-dependent cell-mediated cytotoxicity ("ADCC") is an immune mechanism by which immune effector cells cause the lysis of antibody-coated target cells. Target cells are cells to which antibodies or derivatives containing an Fc domain specifically bind, typically via a protein portion that is N-terminus of the Fc domain. As used herein, the term "decreased ADCC" is defined as either a decrease in the number of target cells lysed at a given time by a given concentration of antibody in the culture medium surrounding the target cells, due to the ADCC mechanism as defined above, and / or an increase in the concentration of antibody in the culture medium surrounding the target cells required to achieve the lysis of a given number of target cells at a given time, due to the ADCC mechanism. ADCC reduction is compared to unmanipulated ADCC mediated by the same antibody produced by the same type of host cells using the same standard methods of production, purification, formulation and storage (known to those skilled in the art). For example, an amino acid substitution that reduces ADCC, such as a reduction in ADCC mediated by an antibody containing its Fc domain, is compared to ADCC mediated by the same antibody that does not contain this amino acid substitution in the Fc domain. Suitable assays for measuring ADCC are well known in the art (see, for example, PCT International Publication No. 2006 / 082515 or No. 2012 / 130831).

[0037] The "class" of an antibody refers to the type of constant domain or constant region held by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. In certain embodiments, the antibody is the IgG1 isotype. In certain embodiments, the antibody is the IgG1 isotype with P329G, L234A, and L235A mutations to reduce the effector function of the Fc region. In other embodiments, the antibody is the IgG2 isotype. In certain embodiments, the antibody is the IgG4 isotype with the S228P mutation in the hinge region to improve the stability of the IgG4 antibody. The heavy chain constant domains corresponding to different classes of immunoglobulins are called α, σ, ε, γ, and μ, respectively. The light chain of an αδεγμ antibody can be assigned to one of two types, called kappa (κ) or lambda (λ), based on the amino acid sequence of its constant domain.

[0038] As used in this application, the terms “human-derived constant region” or “human constant region” refer to the constant heavy chain region and / or constant light chain kappa or lambda region of a human antibody of subclass IgG1, IgG2, IgG3, or IgG4. Such constant regions may be used in human or humanized antibodies, and are publicly known in the art, for example, as described in Kabat, EA, et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991) (see also, for example, Johnson, G., and Wu, TT, Nucleic Acids Res. 28 (2000) 214-218; Kabat, EA, et al., Proc. Natl. Acad. Sci. USA 72 (1975) 2785-2788). Unless otherwise specified herein, the numbering of amino acid residues in the constant region follows the EU numbering system (also known as the Kabat EU index), as described in Kabat, EA et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991), NIH Publication 91-3242.

[0039] A "crossover" Fab molecule (also called "crossFab") refers to a Fab molecule in which the variable domains of the Fab heavy chain and light chain are exchanged (i.e., replaced by each other). Specifically, a crossover Fab molecule includes a peptide chain composed of a light chain variable domain VL and a heavy chain constant domain 1CH1 (VL-CH1, from the N-terminus to the C-terminus), and a peptide chain composed of a heavy chain variable domain VH and a light chain constant domain CL (VH-CL, from the N-terminus to the C-terminus). For clarity, in a crossover Fab molecule in which the variable domains of the Fab light chain and Fab heavy chain are exchanged, the peptide chain containing the heavy chain constant domain 1CH1 is referred to herein as the "heavy chain" of the crossover Fab molecule.

[0040] The “effective amount” of a drug, such as a pharmaceutical composition, refers to the amount that is effective in the dosage and duration required to achieve the desired therapeutic or preventive outcome.

[0041] "Effector function" refers to the biological activity resulting from the Fc region of an antibody, which varies depending on the antibody isotype. Examples of antibody effector functions include C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, downregulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

[0042] As used herein, the terms “to manipulate,” “to be manipulated,” and “to manipulate” are understood to include any manipulation or post-translational modification of the peptide backbone of naturally occurring or recombinant polypeptides or fragments thereof. Manipulation includes modification of amino acid sequences, modification of glycosylation patterns, or modification of the side chain groups of individual amino acids, and combinations thereof.

[0043] As used herein, the terms “first,” “second,” or “third” with respect to Fab molecules, etc., are used for convenience of distinction when there are two or more parts of each type. The use of these terms is not intended to give a particular order or orientation of immunoactivated Fc domain antigen-binding molecules unless expressly indicated so.

[0044] A "Fab molecule" refers to a protein consisting of the VH and CH1 domains of the immunoglobulin heavy chain ("Fab heavy chain") and the VL and CL domains of the immunoglobulin light chain ("Fab light chain").

[0045] "Fused" means that the constituent elements (e.g., the Fab molecule and the Fc domain subunit) are linked either directly by peptide bonds or via one or more peptide linkers.

[0046] As used herein, the term “single-chain” refers to a molecule comprising amino acid monomers linearly linked by peptide bonds. In certain embodiments, one of the antigen-binding moieties is a single-chain Fab molecule, i.e., a Fab molecule in which a Fab light chain and a Fab heavy chain are linked by a peptide linker to form a single peptide chain. In certain such embodiments, the C-terminus of the Fab light chain is connected to the N-terminus of the Fab heavy chain in the single-chain Fab molecule.

[0047] In contrast, a "conventional" Fab molecule refers to a Fab molecule in its natural form, that is, a Fab molecule comprising a heavy chain composed of a variable domain and a constant domain (VH-CH1 in the direction from N to the C-terminus) and a light chain composed of a variable domain and a constant region (VL-CL in the direction from N to the C-terminus).

[0048] The terms "full-length antibody," "intact antibody," and "whole antibody" are used interchangeably herein and refer to antibodies having a structure substantially similar to that of a natural antibody or having a heavy chain containing an Fc region as defined herein.

[0049] In this specification, the terms “Fc domain” or “Fc region” are used to define the C-terminal region of an immunoglobulin heavy chain that includes at least a portion of the constant region. The terms include both the native sequence Fc region and the variant Fc region. The boundary of the Fc region of an IgG heavy chain may vary slightly, but the human IgG heavy chain Fc region is typically defined to extend from Cys226 or Pro230 to the carboxyl terminus of the heavy chain. However, antibodies produced by host cells may undergo post-translational cleavage of one or more amino acids from the C-terminus of the heavy chain, particularly one or two amino acids. Thus, by expression of a particular nucleic acid molecule encoding a full-length heavy chain, antibodies produced by host cells may contain the full-length heavy chain or a cleaved variant of the full-length heavy chain (also referred to herein as a “cleaved variant heavy chain”). This may be the case when the final two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447, numbered according to Kabat's EU index). Therefore, the C-terminal lysine (Lys447) or C-terminal glycine (Gly446) and lysine (K447) of the Fc domain may or may not be present. Unless otherwise specified, the amino acid sequence of the heavy chain containing the Fc domain (or a subunit of the Fc domain as defined herein) is shown herein without the C-terminal glycine-lysine dipeptide. In one embodiment of the present invention, the heavy chain containing the Fc domain subunit identified herein includes a further C-terminal glycine-lysine dipeptide (G446 and K447, numbered according to the Kabat EU index). In another embodiment of the present invention, the heavy chain containing the Fc domain subunit identified herein includes a further C-terminal glycine residue (G446, numbered according to the Kabat EU index). The compositions of the present invention, such as pharmaceutical compositions described herein, comprise a collection of antigen-binding molecules of the present invention. The collection of antigen-binding molecules may include molecules containing full-length heavy chains and molecules containing cleaved variant heavy chains.The antigen-binding molecule assembly may consist of a mixture of molecules having full-length heavy chains and molecules having cleaved variant heavy chains, wherein at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the antigen-binding molecules have cleaved variant heavy chains. In one embodiment of the present invention, a composition comprising the antigen-binding molecule assembly of the present invention comprises an antigen-binding molecule comprising a heavy chain containing a subunit of the Fc domain specified herein, comprising a further C-terminal glycine-lysine dipeptide (G446 and K447, numbered according to the Kabat EU index). In one embodiment of the present invention, a composition comprising the antigen-binding molecule assembly of the present invention comprises an immunoactivating Fc domain-binding molecule comprising a heavy chain containing a subunit of the Fc domain specified herein, comprising a further C-terminal glycine residue (G446, numbered according to the Kabat EU index). In one embodiment of the present invention, such a composition comprises an antigen-binding molecule comprising a molecule comprising a heavy chain comprising a subunit of the Fc domain specified herein, a molecule comprising a heavy chain comprising a subunit of the Fc domain specified herein, comprising a further C-terminal glycine residue (G446, numbered according to Kabat's EU index), and a molecule comprising a heavy chain comprising a subunit of the Fc domain specified herein, comprising a further C-terminal glycine-lysine dipeptide (G446 and K447, numbered according to Kabat's EU index). Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region follows the EU numbering system (also called the EU index), as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991 (see also above). As used herein, a “subunit” of the Fc domain refers to one of the two polypeptides that form the dimeric Fc domain, i.e., a polypeptide containing the C-terminal constant region of an immunoglobulin heavy chain that is capable of stable self-assembly.For example, the subunits of the IgG Fc domain include the IgG CH2 and IgG CH3 constant domains.

[0050] As used herein, "Fc domain binding moiety" is an antigen-binding moiety that can bind to an Fc domain.

[0051] As used herein, "half-life extended Fc" refers to the Fc domain (if present) contained in the immunoactivating Fc domain binding molecule of the present invention. As used herein, "targeted Fc" refers to the Fc domain contained in the targeted antibody of the present invention.

[0052] The terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably and refer to cells into which exogenous nucleic acids have been introduced, and cells including the offspring of such cells. Host cells include “transformed organisms” and “transformed cells,” which include primary transformed cells and their offspring derived from primary transformed cells, regardless of passage number. Offspring may not have exactly the same nucleic acid content as the parent cells and may contain mutations. This specification includes mutant offspring that have the same function or biological activity as those screened or selected in the initially transformed cells.

[0053] An "activated Fc receptor" is an Fc receptor that, after engagement by the Fc domain of an antibody, triggers a signaling event that stimulates receptor-containing cells to exert effector function. Human activated Fc receptors include FcγRIIIa (CD16a), FcγRI (CD64), FcγRIIa (CD32), and FcαRI (CD89).

[0054] A "human antibody" is defined as an antibody produced by a human or human cell, or an antibody having an amino acid sequence corresponding to the amino acids of a non-human antibody derived from the human antibody repertoire or a sequence encoding a human antibody. This definition of a human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues.

[0055] The "Human Consensus Framework" is a framework representing the most commonly occurring amino acid residues in the selection of human immunoglobulin VL or VH framework sequences. Generally, the selection of human immunoglobulin VL or VH sequences is from subgroups of variable domain sequences. Generally, the sequence subgroups are those described in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), vols. 1-3. In one embodiment, for VL, the subgroup is subgroup Kappa I, as described in Kabat et al. In another embodiment, for VH, the subgroup is subgroup Kappa III, as described in Kabat et al.

[0056] A “humanized” antibody refers to a chimeric antibody containing amino acid residues derived from non-human CDRs and amino acid residues derived from human FRs. In certain embodiments, a humanized antibody contains at least one, usually two, variable domains in all or nearly all CDRs corresponding to the variable domains of the non-human antibody, and in all or nearly all FRs corresponding to the variable domains of the human antibody. A humanized antibody may optionally contain at least a portion of the antibody constant region derived from a human antibody. The “humanized form” of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization.

[0057] As used herein, the terms “hypervariable region” or “HVR” refer to regions of an antibody variable domain that are hypervariable in sequence and determine antigen-binding specificity, such as “complementarity-determining regions” (CDRs).

[0058] Generally, an antibody contains six CDRs, three located in the VH (CDR-H1, CDR-H2, CDR-H3) and three located in the VL (CDR-L1, CDR-L2, CDR-L3). Illustrative CDRs as used herein include: (a) Hypervariable loops formed at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)), (b) CDRs present in amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)); and (c) Antigen contact occurring at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) is an example (MacCallum et al. J.Mol.Biol.262:732-745 (1996)).

[0059] Unless otherwise specified, the CDR is determined in accordance with Kabat et al. above. Those skilled in the art will understand that the notation of the CDR may be determined in accordance with Chothia above, McCallum above, or any other scientifically recognized nomenclature system.

[0060] As used herein, “immunoactivating moiety” refers to one or more polypeptides that, upon interaction with an antigen, receptor, or ligand (or other cellular element that induces activation) on an immune cell, induce activation of an immune cell (e.g., a T cell). An example of an immunoactivating moiety is an antigen-binding molecule that can bind to an activated T cell antigen, triggering a signaling cascade of the T cell receptor complex. In certain embodiments, the immunoactivating moiety is an antigen-binding moiety that can bind to CD3, particularly the epsilon subunit of CD3 (see UniProt number P07766 (version 130), NCBI RefSeq number NP_000724.1; or UniProt number Q95LI5 (version 49), NCBI GenBank number BAB71849.1). Other exemplary immunoactivating moieties are antigen-binding moieties that can bind to cytokines described herein (e.g., IL2), costimulatory T cell antigens (e.g., CD28, 4-1BB), or costimulatory ligands (e.g., 4-1BBL).

[0061] An "immunoconjugate" is an antibody conjugated to one or more heterologous molecules, including but not limited to cytotoxic agents.

[0062] The "individual" or "subject" is a mammal. Mammals include, but are not limited to, domestic animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain aspects, the individual or subject is a human.

[0063] An "isolated" antibody is an antibody that has been separated from its natural environment. In some embodiments, antibodies are purified to a purity higher than 95% or 99%, for example, by electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse-phase HPLC). For a review of methods for evaluating antibody purity, see, for example, Flatman et al., J.Chromatogr.B 848:79-87 (2007).

[0064] The term "immunoglobulin molecule" refers to a protein that has the structure of a naturally occurring antibody. For example, IgG class immunoglobulins are heterotetrameric glycoproteins with a weight of approximately 150,000 daltons, composed of two disulfide-linked light chains and two heavy chains. From the N-terminus to the C-terminus, each heavy chain has a variable domain (VH) (also called a variable heavy chain domain or heavy chain variable region) followed by three constant domains (CH1, CH2, and CH3) (also called heavy chain constant regions). Similarly, from the N-terminus to the C-terminus, each light chain has a variable domain (VL, also called the variable light chain domain or light chain variable region) followed by a constant light chain (CL) domain (also called the light chain constant region). The heavy chain of an immunoglobulin may be assigned to one of five types called α(IgA), δ(IgD), ε(IgE), γ(IgG), or μ(IgM), some of which may be further divided into subtypes such as γ1(IgG1), γ2(IgG2), γ3(IgG3), γ4(IgG4), α1(IgA1), and α2(IgA2). The light chain of an immunoglobulin may be assigned to one of two types called kappa (κ) and lambda (λ) based on the amino acid sequence of its constant domain. An immunoglobulin consists of two Fab molecules and an Fc domain, connected via an immunoglobulin hinge region.

[0065] "Framework" or "FR" refers to variable domain residues other than the complementarity-determining region (CDR). The variable domain FR generally consists of four FR domains: FR1, FR2, FR3, and FR4. Therefore, the CDR and FR sequences generally have the following sequence in VH (or VL): FR1-CDR-H1(CDR-L1)-FR2-CDR-H2(CDR-L2)-FR3-CDR-H3(CDR-L3)-FR4.

[0066] "Modifications that promote the association of the first and second subunits of the Fc domain" are manipulations of the peptide backbone or post-translational modifications of the Fc domain subunits that reduce or prevent the association of a polypeptide containing an Fc domain subunit with an identical polypeptide for homodimer formation. When used herein, association-promoting modifications include distinct modifications for each of the two Fc domain subunits that are desired to associate (i.e., the first and second subunits of the Fc domain), and the modifications are complementary to each other in order to promote the association of the two Fc domain subunits. For example, the association-promoting modifications may alter the structure or charge of one or both of the Fc domain subunits so that they perform a sterically or electrostatically desired association. Thus, (hetero)dimerization occurs between a polypeptide containing a first Fc domain subunit and a polypeptide containing a second Fc domain subunit, and these may not be identical in the sense that the further components (e.g., antigen-binding moieties) that fuse to each subunit are not the same. In some embodiments, the association-promoting modifications include amino acid mutations within the Fc domain, specifically amino acid substitutions. In certain embodiments, the modifications that facilitate association include distinct amino acid mutations, specifically amino acid substitutions, in each of the two subunits of the Fc domain.

[0067] The term “monoclonal antibody,” as used herein, refers to an antibody obtained from a substantially homogeneous collection of antibodies. That is, with the exception of possible variant antibodies that are generally present in small amounts, including, for example, naturally occurring mutations or mutations that occur during the manufacture of the monoclonal antibody preparation, the individual antibodies constituting the collection are identical and / or bind to the same epitope. In contrast to polyclonal antibody preparations, which typically contain various antibodies against various determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is against a single determinant on an antigen. Therefore, the modifier “monoclonal” indicates the characteristic of an antibody obtained from a substantially homogeneous collection of antibodies and should not be interpreted as requiring antibody production by any particular method. For example, monoclonal antibodies according to the present invention can be produced by a variety of techniques, including but not limited to hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of a human immunoglobulin locus, and such methods and other exemplary methods for producing monoclonal antibodies described herein are described herein.

[0068] A "naked antibody" refers to an antibody that is not bound to a heterogeneous site (e.g., a cytotoxic site) or a radioactive label. Naked antibodies may be present in a pharmaceutical composition.

[0069] "Natural antibodies" refer to naturally occurring immunoglobulin molecules with various structures. For example, a natural IgG antibody is a heterotetrameric glycoprotein with approximately 150,000 daltons, containing two identical light chains and two identical heavy chains linked by disulfide bonds. From the N-terminus to the C-terminus, each heavy chain has a variable domain (VH), also called a variable heavy domain or heavy chain variable region, followed by three constant heavy domains (CH1, CH2, and CH3). Similarly, from the N-terminus to the C-terminus, each light chain has a variable domain (VL), also called a variable light domain or light chain variable region, followed by a constant light (CL) domain.

[0070] The terms “nucleic acid molecule” or “polynucleotide” include any compound and / or substance containing polymers of nucleotides. Each nucleotide is composed of a base, specifically a purine or pyrimidine base (i.e., cytosine (C), guanine (G), adenine (A), thymine (T), or uracil (U)), a sugar (i.e., deoxyribose or ribose), and a phosphate group. Often, nucleic acid molecules are described by a base sequence, where the bases represent the primary structure (linear structure) of the nucleic acid molecule. The base sequence is typically represented 5' to 3'. In this specification, the term nucleic acid molecule includes deoxyribonucleic acid (DNA), e.g., complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), in particular messenger RNA (mRNA), synthetic forms of DNA or RNA, and mixed polymers containing two or more of these molecules. Nucleic acid molecules may be linear or cyclic. In addition, the term nucleic acid molecule includes both sense strands and antisense strands, as well as both single-stranded and double-stranded forms. Furthermore, nucleic acid molecules described herein may include naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides, including derivatized sugar or phosphate backbone links or chemically modified residues, include modified nucleotide bases. Nucleic acid molecules also include DNA and RNA molecules suitable as vectors for the direct expression of the antibodies of the present invention in vitro and / or in vivo, for example, in a host or patient. Such DNA (e.g., cDNA) or RNA (e.g., mRNA) vectors may be unmodified or modified. For example, mRNA may be chemically modified to enhance the stability of the RNA vector and / or the expression of the encoded molecule, so that mRNA can be injected into a subject to produce an antibody in vivo. (For example, see Stadler et al., Nature Medicine 2017, published online 12 June 2017, doi:10.1038 / nm.4356 or European Patent No. 2 101823B1.)

[0071] A nucleic acid or polynucleotide having a nucleotide sequence that is at least, for example, 95% "identical" to a reference nucleotide sequence of the present invention is intended to be identical to the reference nucleotide sequence, except that the nucleotide sequence of the polynucleotide may contain up to 5 point mutations per 100 nucleotides of the reference nucleotide sequence. In other words, to obtain a polynucleotide having a nucleotide sequence that is at least 95% identical to the reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or substituted with other nucleotides, or up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. Such modifications to the reference sequence may occur at the 5' or 3' terminal position of the reference nucleotide sequence, or at any position between these terminal positions, and may be individually scattered among the residues in the reference sequence, or may be scattered as one or more consecutive groups within the reference sequence. In practice, whether any particular polynucleotide sequence is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of the present invention can be conventionally determined using a known computer program, such as the one described above for polypeptides (e.g., ALIGN-2).

[0072] The term "expression cassette" refers to a polynucleotide produced by recombination or synthesis, comprising a specific set of nucleic acid elements capable of transcribing a particular nucleic acid within a target cell. Recombinant expression cassettes can be incorporated into plasmids, chromosomes, mitochondrial DNA, plastid DNA, viruses, or nucleic acid fragments. Typically, the recombinant expression cassette portion of an expression vector includes, in particular, the nucleic acid sequence to be transcribed and a promoter. In certain embodiments, the expression cassette of the present invention comprises a polynucleotide sequence or fragment thereof encoding the bispecific antigen-binding molecule of the present invention.

[0073] Percent (%) amino acid sequence identity with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to those in the reference polypeptide sequence, after aligning the sequences for alignment purposes and introducing gaps if necessary to achieve the highest possible sequence identity, without considering any conservative substitutions as part of the sequence identity. Alignment for determining percent amino acid sequence identity can be achieved using various methods within the scope of the art, such as publicly available computer software like BLAST, BLAST-2, Clustal W, Megalign (DNASTAR) software, or FASTA program packages. Those skilled in the art can determine appropriate parameters for aligning the sequences, including any algorithm required to achieve the highest degree of alignment over the entire length of the sequences being compared. Alternatively, the percentage identity value can be generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was created by Genentech, Inc., and its source code is filed in the user documentation of the U.S. Copyright Office (Washington DC, 20559), registered under U.S. Copyright Registration No. TXU510087, and published internationally in publication No. 2001 / 007611.

[0074] However, for the purposes of this specification, the percentage amino acid sequence identity values ​​are generated using the ggsearch program in FASTA package version 36.3.8c, or subsequently using the BLOSUM50 comparison matrix. The FASTA program package is certified by WRPearson and DJLipman (1988), "Improved Tools for Biological Sequence Analysis," PNAS 85:2444-2448; WRPearson (1996), "Effective protein sequence comparison," Meth.Enzymol. 266:227-258; and Pearson et al. (1997), Genomics 46:24-36, and is publicly available at www.fasta.bioch.virginia.edu / fasta_www2 / fasta_down.shtml or www.ebi.ac.uk / Tools / sss / fasta. Alternatively, the ggsearch(global protein:protein) program and its default options (BLOSUM50;open:-10;ext:-2;Ktup=2) can be used to compare sequences using a public server accessible at fasta.bioch.virginia.edu / fasta_www2 / index.cgi, ensuring that a global rather than local alignment is performed. The amino acid identity percentage is given in the output alignment header. As used herein, the term “polypeptide” refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term “polypeptide” refers to any chain of two or more amino acids and does not refer to a product of a specific length. Thus, peptide, dipeptide, tripeptide, oligopeptide, “protein,” “amino acid chain,” or any other term used to refer to a chain of two or more amino acids are included in the definition of “polypeptide,” and the term “polypeptide” may be used in place of or interchangeably with any of these terms.The term "polypeptide" is intended to refer to the products of post-expression modifications of polypeptides, including, but not limited to, glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, cleavage by proteolysis, or modification with amino acids not naturally occurring. Polypeptides may be derived from natural biosources or produced by recombinant technology, but are not necessarily translated from a specified nucleic acid sequence. Polypeptides may arise in any manner, including by chemical synthesis. Polypeptides of the present invention may be in size of about 3 or more, 5 or more, 10 or more, 20 or more, 25 or more, 50 or more, 75 or more, 100 or more, 200 or more, 500 or more, 1,000 or more, or 2,000 or more amino acids. Polypeptides may have a defined three-dimensional structure, but polypeptides do not necessarily have such a structure. Polypeptides having a defined three-dimensional structure are called folded, and polypeptides that do not have a defined three-dimensional structure but can rather adopt many different conformations are called unfolded.

[0075] The terms "pharmaceutical composition" or "pharmaceutical preparation" refer to a preparation that is in a form that enables the effective biological activity of the active ingredient contained herein, and that does not contain any additional components that are unacceptably toxic to the subject to which the pharmaceutical composition is administered.

[0076] A "pharmaceutically acceptable carrier" refers to a component in a pharmaceutical composition or preparation other than the active ingredient, which is non-toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0077] The term “package insert” is used to refer to the instructions typically included in the commercial packaging of a therapeutic product, which contain information about the indications, usage, dosage, administration, combination therapy, contraindications, and / or warnings regarding the use of such therapeutic product.

[0078] "Decreased binding," for example, decreased binding to the Fc receptor, refers to a reduction in affinity for each interaction, as measured, for example, by SPR. For clarity, this term also includes a reduction in affinity to zero (or below the detection limit of the analytical method), i.e., complete termination of the interaction. Conversely, "increased binding" refers to an increase in binding affinity for individual interactions.

[0079] "Specific binding" means that the binding is antigen-selective and can be distinguished from undesirable or nonspecific interactions. The ability of an antigen-binding moiety to bind to a specific antigenic determinant can be measured by enzyme-linked immunosorbent assay (ELISA) or other techniques known to those skilled in the art, such as surface plasmon resonance (SPR) (analyzed with a BIAcore instrument) (Liljeblad et al., Glyco J 17, 323-329 (2000)), and conventional binding assays (Heeley, Endocr Res 28, 217-229 (2002)). In one embodiment, the degree of binding of an antigen-binding moiety to an unrelated protein is less than about 10% of the binding of the antigen-binding moiety to an antigen, for example, as measured by SPR. In certain embodiments, an antigen-binding moiety that binds to an antigen, or an antigen-binding molecule containing this antigen-binding moiety, has a dissociation constant (K D ) is ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (for example, 10 -8 M or less, for example, 10 -8 M~10 -13 M, for example, 10 -9 M~10 -13 M) is the answer.

[0080] As used herein, “T cell activation” refers to one or more cellular responses of T lymphocytes, particularly cytotoxic T lymphocytes, selected from proliferation, differentiation, cytokine secretion, release of cytotoxic effector molecules, cytotoxic activity, and expression of activation markers. The immunoactivating Fc domain-binding molecules of the present invention can induce T cell activation. Suitable assays for measuring T cell activation are known in the art and are described herein.

[0081] As used herein, “target cell antigen” means an antigenic determinant presented on the surface of a target cell, such as a cancer cell or a tumor cell, such as a tumor stromal cell. In certain embodiments, the target cell antigen is CD20, in particular human CD20 (see UniProt number P11836).

[0082] The “therapeutic effective dose” of a drug, such as a pharmaceutical composition, refers to the effective amount in the dose and duration required to obtain the desired therapeutic or prophylactic outcome. The therapeutic effective dose of a drug is effective in eliminating, reducing, delaying, minimizing, or preventing the adverse effects of a disease, for example, by eliminating, reducing, delaying, minimizing, or preventing them.

[0083] As used herein, “treatment” (and its grammatical variations, e.g., “to treat” or “treating”) refers to a clinical intervention in an attempt to alter the natural course of a disease in the treated individual, and may be performed for preventive purposes or during the course of a clinicopathological disease. Desired effects of treatment include, but are not limited to, preventing the onset or recurrence of the disease, reducing symptoms, attenuating any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, achieving remission or mitigation of symptoms, and achieving recovery or improving prognosis. In some embodiments, the antibodies of the present invention are used to delay the onset of a disease or to slow the progression of a disease.

[0084] As used herein, the term "valence" refers to the presence of a specific number of antigen-binding sites within an antigen-binding molecule. Therefore, the term "monovalent binding to an antigen" refers to the presence of one (and fewer than one) antigen-specific antigen-binding sites within an antigen-binding molecule.

[0085] The term "variable region" or "variable domain" refers to a domain in the heavy or light chain of an antibody that is involved in the binding of the antibody to the antigen. The variable domains of the heavy and light chains of natural antibodies (VH and VL, respectively) generally have similar structures, and each domain contains four conserved framework regions (FRs) and three complementarity-determining regions (CDRs). For example, Kindt et al., Kuby Immunology, 6. th See WH Freeman and Co., page 91 (2007). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind to a specific antigen may be isolated by screening a library of complementary VL or VH domains using the VH or VL domain of the antibody that binds to the antigen, respectively. See, for example, Portolano et al., J.Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0086] As used herein, the term “vector” refers to a nucleic acid molecule capable of replicating another nucleic acid to which it is linked. This term includes vectors as self-replicating nucleic acid structures, and vectors incorporated into the genome of a host cell into which they are introduced. Certain vectors can direct the expression of the nucleic acid to which they are functionally linked. Such vectors are referred to herein as “expression vectors.”

[0087] As used herein, the term "interleukin-2" or "IL-2" refers to any native IL-2 derived from any vertebrate source, including, but not limited to, mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise indicated. This term encompasses both untreated IL-2 and any form of IL-2 resulting from the processing of cells. This term also includes naturally occurring variants of IL-2, such as splice variants or allelic variants. The amino acid sequence of exemplary human IL-2 is shown in SEQ ID NO: 166. Untreated human IL-2 contains an N-terminal 20 amino acid signal peptide that is not present in the mature IL-2 molecule.

[0088] As used herein, the term "IL-2 variant" or "variant IL-2 polypeptide" is intended to encompass any variant form of the IL-2 molecule, including full-length IL-2, a truncated form of IL-2, or a form in which IL-2 is linked to another molecule by fusion or chemical conjugation, etc. When used in reference to IL-2, "full-length" is intended to mean the mature native-length IL-2 molecule. For example, full-length human IL-2 refers to a molecule containing 133 amino acids (e.g., SEQ ID NO: 166). The various forms of IL-2 variants are characterized by having at least one amino acid mutation that affects the interaction between IL-2 and CD25. This mutation may include substitution, deletion, truncation, or modification of the wild-type amino acid residue normally located at that position. Variants obtained by amino acid substitution are preferred. Unless otherwise indicated, IL-2 variants may be referred to herein as variant IL-2 peptide sequences, variant IL-2 polypeptides, variant IL-2 proteins, or variant IL-2 analogs.

[0089] The naming of the various forms of IL-2 is done herein with respect to the sequence shown in SEQ ID NO: 19. Various names may be used herein to indicate the same mutation. For example, the mutation from phenylalanine to alanine at position 42 can be shown as 42A, A42, A 42 , F42A or Phe42Ala.

[0090] As used herein, "human IL-2 molecule" means an IL-2 molecule containing an amino acid sequence that is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, or at least about 96% identical to the human IL-2 sequence of Sequence ID No. 166. In particular, sequence identity is at least about 95%, and more particularly, at least about 96%. In certain embodiments, the human IL-2 molecule is a full-length IL-2 molecule.

[0091] As used herein, the terms “CD25” or “α-subunit of the IL-2 receptor” refer to any natural CD25 from any vertebrate source, including mammals, e.g., primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise specified. The term encompasses “full-length” untreated CD25 and any form of CD25 resulting from cell processing. The term also encompasses naturally occurring variants of CD25, e.g., splice variants or allele variants. In certain embodiments, CD25 is human CD25. The amino acid sequence of human CD25 can be found, for example, in UniProt entry number P01589 (version 185).

[0092] As used herein, the term "high affinity IL-2 receptor" refers to the receptor γ-subunit (common cytokine receptor γ-subunit, γ cThis refers to a heterotrimer form of the IL-2 receptor consisting of the γ-subunit and β-subunit (see CD132, UniProt entry number P14784 (version 192)), the receptor β-subunit (also known as CD122 or p70, see UniProt entry number P31785 (version 197)), and the receptor α-subunit (also known as CD25 or p55, see UniProt entry number P01589 (version 185)). The term "moderate affinity IL-2 receptor," in contrast, refers to an IL-2 receptor that contains only the γ-subunit and β-subunit and does not contain the α-subunit (see, for example, Olejniczak and Kasprzak, Med Sci Monit 14, RA179-189 (2008)).

[0093] The terms "TNF ligand family members" or "TNF family ligands" refer to pro-inflammatory cytokines. Cytokines, in general, and especially members of the TNF ligand family, play important roles in stimulating and regulating the immune system. Currently, 19 cytokines have been identified as members of the TNF (tumor necrosis factor) ligand superfamily based on similarities in sequence, function, and structure. All of these ligands are type II transmembrane proteins with a C-terminal extracellular domain (external domain), an N-terminal intracellular domain, and a single transmembrane domain. The C-terminal extracellular domain, known as the TNF homologous domain (THD), has 20-30% amino acid identity among superfamily members and is responsible for receptor binding. The TNF external domain also carries the TNF ligand, which forms a trimer complex recognized by its specific receptor. Members of the TNF ligand family include lymphotoxin α (also known as LTA or TNFSF1), TNF (also known as TNFSF2), LTβ (also known as TNFSF3), OX40L (also known as TNFSF4), CD40L (also known as CD154 or TNFSF5), FasL (also known as CD95L, CD178 or TNFSF6), CD27L (also known as CD70 or TNFSF7), CD30L (also known as CD153 or TNFSF8), 4-1BBL (also known as TNFSF9), and TRAIL (APO2L, CD253 or TNFSF1). The group is selected from the following: 0 (also known as 0), RANKL (also known as CD254 or TNFSF11), TWEAK (also known as TNFSF12), APRIL (also known as CD256 or TNFSF13), BAFF (also known as CD257 or TNFSF13B), LIGHT (also known as CD258 or TNFSF14), TL1A (also known as VEGI or TNFSF15), GITRL (also known as TNFSF18), EDA-A1 (also known as ectodiplassin A1), and EDA-A2 (also known as ectodiplassin A2).Unless otherwise specified, this term refers to natural TNF family ligands derived from any vertebrate source, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus macaques), and rodents (e.g., mice and rats).

[0094] The term "co-stimulatory TNF ligand family members," or "co-stimulatory TNF family ligands," refers to a subgroup of TNF ligand family members that can co-stimulate T cell proliferation and cytokine production. These TNF family ligands can co-stimulate TCR signaling when they interact with their corresponding TNF receptors, and this interaction with their receptors leads to the recruitment of TNFR-related factors (TRAFs) that initiate a signaling cascade resulting in T cell activation. Co-stimulatory TNF family ligands are selected from the group consisting of 4-1BBL, OX40L, GITRL, CD70, CD30L, and LIGHT, and more specifically, the co-stimulatory TNF ligand family member is 4-1BBL.

[0095] As previously described herein, 4-1BBL is a type II transmembrane protein and a member of the TNF ligand family. Complete or full-length 4-1BBL having the amino acid sequence of SEQ ID NO: 69 has been described as forming trimers on the cell surface. Trimerization is enabled by a specific motif in the extracellular domain of 4-1BBL, which is referred to herein as the “trimerization region.” Amino acids 50–254 of the human 4-1BBL sequence form the extracellular domain of 4-1BBL, but fragments of this domain can also form trimers. In certain embodiments of the present invention, the term "external domain of 4-1BBL or fragment thereof" refers not only to polypeptides having amino acid sequences selected from SEQ ID NO: 120 (amino acids 52-254 of human 4-1BBL), SEQ ID NO: 117 (amino acids 71-254 of human 4-1BBL), SEQ ID NO: 119 (amino acids 80-254 of human 4-1BBL), and SEQ ID NO: 118 (amino acids 85-254 of human 4-1BBL), or polypeptides having amino acid sequences selected from SEQ ID NO: 121 (amino acids 71-248 of human 4-1BBL), SEQ ID NO: 124 (amino acids 52-248 of human 4-1BBL), SEQ ID NO: 123 (amino acids 80-248 of human 4-1BBL), and SEQ ID NO: 122 (amino acids 85-248 of human 4-1BBL), but also to other fragments of trimerizable external domains.

[0096] The “extracellular domain” is a domain of a membrane protein that extends into the extracellular space (i.e., the space outside the target cell). The extracellular domain is typically the portion of the protein that initiates contact with the surface, resulting in signal transduction. Thus, the extracellular domain of a TNF ligand family member as defined herein refers to the portion of the TNF ligand protein that extends into the extracellular space (extracellular domain), but also includes shorter portions or fragments thereof that are responsible for trimerization and binding to the corresponding TNF receptor. Therefore, the term “extracellular domain or fragment thereof of a TNF ligand family member” refers to the extracellular domain of a TNF ligand family member that forms the extracellular domain, or the portion thereof that can still bind to the receptor (receptor-binding domain).

[0097] As used herein, “PD1,” “human PD1,” “PD-1” or “human PD-1” (also known as programmed death protein 1 or programmed death 1) refers to the human protein PD1. See also UniProt entry number Q15116 (version 156). As used herein, “binding to PD-1,” “specifically binding to PD-1,” “that binds to PD-1,” or antibody or “anti-PD-1 antibody” refers to an antibody capable of binding to PD-1, particularly to the PD-1 polypeptide expressed on the cell surface, with sufficient affinity to be useful as a diagnostic and / or therapeutic agent when targeting PD-1. In one embodiment, the degree of binding of an anti-PD-1 antibody to unrelated non-PD-1 proteins is less than approximately 10% of the binding of the antibody to PD-1, as measured, for example, by radioimmunoassay (RIA) or flow cytometry (FACS), or by surface plasmon resonance assay using a biosensor system such as the Biacore® system. In a particular embodiment, the antibody that binds to PD-1 has a KD value of binding affinity to human PD-1 of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g., 10 -8 M or less, for example, 10 -8 M~10 -13 M, for example 10 -9 M~10 -13 M) In one embodiment, the binding affinity KD value is determined in a surface plasmon resonance assay using the extracellular domain (ECD) of human PD-1 as the antigen.

[0098] Detailed description of the invention The present invention provides a modular antibody-based platform for flexible antigen targeting and individual immune cell stimulation that can be adapted to desired indications. Compared to conventional bispecificity formats and checkpoint modulators that directly engage with their target of interest, the present invention consists of two components that can be individually adapted and used in a plug-and-play manner. This modular platform focuses primarily on two parts: (i) easily produced targeting molecules that have the ability to stimulate immune cells if desired, and (ii) targeted antibodies for precise and selective antigen targeting via an immunoactivating (Fc domain binding) molecule that specifically recognizes the Fc portion of the targeted antibody, thereby recruiting and activating immune effector cells by establishing an immune synapse that redirects, for example, CTLs and subsequently initiates the lysis of the target cell (see Figures 1 and 43). The combination of targeted antibodies and immunoactivating (Fc domain binding) molecules enables a personalized, customizable, ready-made approach to stimulating individual immune cells without requiring the generation of different effector molecules for each unique surface antigen.

[0099] Therefore, in one embodiment, the present invention provides an immunoactivating fragment crystallizable (Fc) domain binding molecule.

[0100] In one aspect of the present invention, an immunoactivating fragment crystallizable (Fc) domain binding molecule, (a) Fc domain binding portion, (b) Immune activation portion and An immune-activating Fc domain-binding molecule is provided, which includes this component.

[0101] In some embodiments, for example, when a short half-life of the immune-activating Fc domain-binding molecule is preferred, the immune-activating Fc domain-binding molecule does not contain an Fc domain. Therefore, the present invention provides an immune-activating Fc domain-binding molecule lacking an Fc domain (see Figures 2O-2Z for exemplary formats).

[0102] However, in many cases, it would be preferable to include an Fc domain in the immunoactivating Fc domain binding molecule of the present invention. The Fc domain confers desirable pharmacokinetic properties to the antibody, including a long serum half-life that contributes to good accumulation in target tissues, and a favorable tissue-to-blood distribution ratio.

[0103] Therefore, in a preferred embodiment of the present invention, (c) an immunoactivating fragment crystallizable (Fc) domain-binding molecule containing a half-life extension Fc domain is provided. In particular, it may be desirable to ensure that the Fc domain binding portion cannot bind to the half-life extension Fc. Binding of the Fc domain binding portion to the half-life extension Fc domain may result in self-binding of the immunoactivating Fc domain-binding molecule, i.e., one immunoactivating Fc domain-binding molecule binds to another (identical) Fc domain-binding molecule via the half-life extension Fc domain. Self-binding may result in cross-linking of multiple immunoactivating Fc domain-binding molecules, which may be undesirable.

[0104] Therefore, in a preferred embodiment of the present invention, an immunoactivating fragment crystallizable (Fc) domain binding molecule is provided, (a) an Fc domain binding moiety that specifically binds to a target Fc domain containing at least one amino acid substitution of the first set, (b) The immune activation portion, (c) Half-life extension Fc and Includes, An immunoactivating fragment crystallizable (Fc) domain binding molecule is provided, in which the Fc domain binding portion does not specifically bind to the half-life extension Fc domain.

[0105] Since the Fc domain binding portion does not specifically bind to the half-life extension Fc domain, self-binding or cross-linking does not occur; that is, the immunoactivating Fc domain binding molecule recognizes and binds only to Fc domains containing at least one amino acid substitution of the first set. Fc domains containing at least one amino acid substitution of the first set are referred to herein as target Fc domains. Fc domains included in the immunoactivating Fc domain binding molecule are referred to herein as half-life extension Fc domains. While not bound by theory, it will be apparent to those skilled in the art that target Fc domains can also extend the half-life of targeted antibodies. However, to clearly distinguish between target Fc domains and Fc domains included in the immunoactivating Fc domain binding molecule of the present invention, the half-life extension Fc domain described herein always refers to an Fc domain included in the immunoactivating Fc domain binding molecule.

[0106] The Fc domains described herein (e.g., target Fc domains or half-life extension Fc domains) consist of a pair of polypeptide chains containing the heavy chain domains of an immunoglobulin molecule. For example, the Fc domain of an immunoglobulin G (IgG) molecule is a dimer, and each subunit contains the CH2 and CH3 IgG heavy chain constant domains. The two subunits of the Fc domain can stably associate with each other. In one embodiment, the immunoactivating Fc domain binding molecule of the present invention contains one or fewer Fc domains.

[0107] As previously stated herein, the Fc domain confers desirable pharmacokinetic properties to antibodies, including a long serum half-life. However, this can simultaneously lead to undesirable targeting of cells expressing the Fc receptor rather than the desired antigen-containing cells. Furthermore, co-activation of the Fc receptor signaling pathway, combined with the T cell activation properties and the long half-life of the immunoactivating Fc domain binding molecule, can result in excessive activation of cytokine receptors and cytokine release leading to severe side effects upon systemic administration. Activation of immune cells other than T cells (those possessing the Fc receptor) may even reduce the effectiveness of the immunoactivating Fc domain binding molecule, for example, due to the potential destruction of T cells by NK cells.

[0108] As described herein, in preferred embodiments, the target Fc domain comprises at least one amino acid substitution of a first set. In one embodiment, at least one amino acid substitution of the first set reduces binding to the Fc receptor and / or reduces effector function. Furthermore, in embodiments in which the immunoactivating Fc domain binding molecule comprises a half-life extension Fc domain, the half-life extension Fc domain may comprise at least one amino acid substitution of a second set. In one embodiment, at least one amino acid substitution of the second set reduces binding to the Fc receptor and / or reduces effector function.

[0109] Accordingly, one particular aspect of the present invention is to reduce the effector function of targeted antibodies and / or immunoactivating antibodies. In such embodiments, the Fc domain binding moiety specifically binds to an Fc domain (target Fc domain) containing at least one amino acid substitution of a first set, but does not specifically bind to an Fc domain (half-life extended Fc domain) containing at least one amino acid substitution of a second set. Fc domain binding moieties having such desirable specificity are described below herein, as are methods for generating further Fc domain binding moieties having desired specificity (e.g., immunization of the mammalian immune system using an Fc domain containing at least one amino acid substitution of a first set, and screening of Fc domain binding moieties that do not specifically bind to an Fc domain containing at least one amino acid substitution of a second set, wherein at least one amino acid substitution of the first and / or second set reduces binding to the Fc receptor and / or reduces effector function).

[0110] Exemplary Fc domain binding moieties that specifically bind to the target Fc domain (where at least one amino acid substitution in the first set includes a P329G substitution) rather than the half-life extension Fc domain (where at least one amino acid substitution in the second set does not include a P329G substitution, i.e., is wild-type at position P329 or includes a terminal amino acid substitution at position P329 other than glycine) are the anti-P329G(M-1.7.24)huIgG1 binder, comprising the CDR sequences of Sequence ID No. 1, 2, 3, 4, 5 and 6 (numbering according to Kabat's EU index), further described in International Publication No. 2017 / 072210.

[0111] Another exemplary Fc domain binding moiety that specifically binds to the target Fc domain rather than the half-life extension Fc domain is the anti-AAA binder containing the CDR sequences SEQ ID NOs: 168, 169, 170, 171, 172, and 173 (numbering according to Kabat's EU index), further described in International Publication No. 2017 / 072210.

[0112] Targeted Fc domains and / or half-life-extending Fc domains may preferably confer increased effector function to targeted antibodies and / or immunoactivating Fc domain-binding molecules, respectively. Therefore, in one embodiment, at least one amino acid substitution in a first set increases binding to the Fc receptor and / or increases effector function. In one embodiment, at least one amino acid substitution in a second set increases binding to the Fc receptor and / or increases effector function. Such Fc domain-binding moieties with desirable specificity can be generated, as described herein, for example, by immunizing the mammalian immune system with an Fc domain containing at least one amino acid substitution in a first set and screening for Fc domain-binding moieties that do not specifically bind to the Fc domain containing at least one amino acid substitution in a second set, wherein at least one amino acid substitution in the first and / or second sets increases binding to the Fc receptor and / or increases effector function.

[0113] Fc mutations (e.g., amino acid substitutions) that confer such binding and / or effector function to the Fc receptor are known in the Art and are described below herein. In one embodiment, the target Fc domain and / or half-life extension Fc domain is an IgG1 or IgG4Fc domain. In one embodiment, the target Fc domain and / or half-life extension Fc domain exhibit reduced binding affinity to the Fc receptor and / or reduced effector function compared to the natural IgG1Fc domain. In such an embodiment, the target Fc domain and / or half-life extension Fc domain (or the molecule containing the Fc domain) individually exhibit less than 50%, preferably less than 20%, more preferably less than 10%, and most preferably less than 5% binding affinity to the Fc receptor compared to the natural IgG1Fc domain (or the molecule containing the natural IgG1Fc domain), and / or less than 50%, preferably less than 20%, more preferably less than 10%, and most preferably less than 5% effector function compared to the natural IgG1Fc domain (or the molecule containing the natural IgG1Fc domain). In one embodiment, the target Fc domain and / or the half-life extension Fc domain (or the molecule containing said Fc domain) does not substantially bind to the Fc receptor and / or induce effector function. In a particular embodiment, the Fc receptor is the Fcγ receptor. In one embodiment, the Fc receptor is the human Fc receptor. In one embodiment, the Fc receptor is the activated Fc receptor. In a specific embodiment, the Fc receptor is the activated human Fcγ receptor, more specifically, the human FcγRIIIa, FcγRI, or FcγRIIa, and most specifically, the human FcγRIIIa. In one embodiment, the effector function is one or more selected from the group consisting of CDC, ADCC, ADCP, and cytokine secretion. In a particular embodiment, the effector function is ADCC. In one embodiment, the target Fc domain and / or the half-life extension Fc domain each exhibit substantially similar binding affinity to the neonatal Fc receptor (FcRn) compared to the natural IgG1 Fc domain.If the target Fc domain and / or the half-life extension Fc domain (or the molecule containing the Fc domain) individually exhibit a binding affinity of more than 70%, particularly more than 80%, and more particularly more than 90% of the binding affinity of the natural IgG1Fc domain (or the molecule containing the natural IgG1Fc domain) to FcRn, substantially similar binding to FcRn is achieved.

[0114] In certain embodiments, the target Fc domain and / or half-life extension Fc domain are individually manipulated to have reduced binding affinity to the Fc receptor and / or reduced effector function compared to the unmanipulated Fc domain. In certain embodiments, the target Fc domain and / or half-life extension Fc domain each contain one or more amino acid substitutions that reduce the binding affinity and / or effector function of the Fc domain to the Fc receptor. Typically, the same one or more amino acid substitutions are present in each of the two subunits of the target Fc domain and / or in each of the two subunits of the half-life extension Fc domain. However, the amino acid substitutions in the target Fc domain and the amino acid substitutions in the half-life extension Fc domain cannot be identical if the non-binding of the Fc domain binding portion to the half-life extension Fc domain should be guaranteed. In such embodiments, it is assumed that at least one amino acid substitution in a first set and at least one amino acid substitution in a second set each contain at least one amino acid substitution that reduces binding to the Fc receptor and / or effector function, as described below herein. In one embodiment, an amino acid substitution reduces the binding affinity of the Fc domain to the Fc receptor. In one embodiment, an amino acid substitution reduces the binding affinity of the Fc domain to the Fc receptor by at least half, at least one-fifth, or at least one-tenth. In embodiments where there are two or more amino acid substitutions that reduce the binding affinity of the target Fc domain to the Fc receptor and / or the half-life-extending Fc domain, the combination of these amino acid substitutions may reduce the binding affinity of the Fc domain to the Fc receptor by at least 10-fold, at least 20-fold, or at least 50-fold. In one embodiment, the targeted antibody and / or immunoactivated Fc domain binding molecule each contains an engineered Fc domain that exhibits a binding affinity of less than 20%, particularly less than 10%, and more particularly less than 5% to the Fc receptor compared to a molecule containing an unengineered Fc domain. In certain embodiments, the Fc receptor is an Fcγ receptor. In some embodiments, the Fc receptor is a human Fc receptor. In some embodiments, the Fc receptor is an activated Fc receptor.In specific embodiments, the Fc receptor is an activated human Fcγ receptor, more specifically human FcγRIIIa, FcγRI, or FcγRIIa, and most specifically human FcγRIIIa. Preferably, binding to each of these receptors is reduced. In some embodiments, binding affinity to complementary components (specifically binding affinity to C1q) is also reduced. In one embodiment, binding affinity to the neonatal Fc receptor (FcRn) is not reduced. Substantially similar binding to FcRn, i.e., preservation of the binding affinity of the Fc domain to the receptor, is achieved when the Fc domain (or a molecule containing the Fc domain) exhibits a binding affinity to FcRn of more than about 70% of that of the unmanipulated form of the Fc domain (or a molecule containing the unmanipulated form of the Fc domain). Targeted Fc domains and / or half-life extended Fc domains, or molecules of the present invention containing the Fc domain, may individually exhibit more than about 80%, and even more than about 90%, of such affinity. In certain embodiments, the target Fc domain and / or half-life extension Fc domain are individually engineered to have reduced effector function compared to the unengineered Fc domain. Reduced effector function may include, but is not limited to, one or more of the following: reduced complement-dependent cytotoxicity (CDC), reduced antibody-dependent cell-mediated cytotoxicity (ADCC), reduced antibody-dependent phagocytosis (ADCP), reduced cytokine secretion, reduced immune complex-mediated antigen uptake by antigen-presenting cells, reduced binding to NK cells, reduced binding to macrophages, reduced binding to monocytes, reduced binding to polymorphonuclear cells, reduced direct signaling-induced apoptosis, reduced cross-linking with target-binding antibodies, reduced dendritic cell maturation, or reduced T cell priming. In one embodiment, the reduced effector function is one or more selected from the group of reduced CDC, reduced ADCC, reduced ADCP, and reduced cytokine secretion. In certain embodiments, the reduced effector function is reduced ADCC. In one embodiment, the reduction in ADCC is less than 20% of the ADCC induced by an unmodified Fc domain (or a molecule containing an unmodified Fc domain).

[0115] At least one amino acid substitution in the first set At least one amino acid substitution of the first set is included in the targeted antibody (target Fc domain), as shown in Figure 1. Thus, in one aspect of the present invention, the target Fc domain described herein includes at least one amino acid substitution of the first set. In one embodiment, at least one amino acid substitution of the first set includes at least one amino acid substitution that reduces the binding affinity and / or effector function of the target Fc domain to the Fc receptor. In one embodiment, the target Fc domain includes an amino acid substitution at a position selected from the group E233, L234, L235, N297, P331 and P329 (numbering according to the Kabat EU index). In a more specific embodiment, the target Fc domain includes an amino acid substitution at a position selected from the group L234, L235 and P329 (numbering according to the Kabat EU index). In some embodiments, the target Fc domain includes amino acid substitutions L234A and L235A (numbering according to the Kabat EU index). In one such embodiment, the target Fc domain is an IgG1Fc domain, particularly a human IgG1Fc domain. In one embodiment, the target Fc domain includes an amino acid substitution at position P329. In a more specific embodiment, the amino acid substitution is P329A or P329G, particularly P329G (numbering according to Kabat's EU index). In one embodiment, the target Fc domain includes an amino acid substitution at position P329 and includes further amino acid substitutions at positions selected from E233, L234, L235, N297 and P331 (numbering according to Kabat's EU index). In a more specific embodiment, the further amino acid substitutions are E233P, L234A, L235A, L235E, N297A, N297D or P331S. In a particular embodiment, the target Fc domain includes amino acid substitutions at positions P329, L234 and L235 (numbering according to Kabat's EU index). In more specific embodiments, the target Fc domain includes amino acid substitutions L234A, L235A, and P329G ("P329G LALA"). In one such embodiment, the target Fc domain is an IgG1Fc domain, particularly a human IgG1Fc domain.The amino acid substitution combination "P329G LALA" almost completely eliminates the Fcγ receptor (and complement) binding of the human IgIgG1Fc domain, as described in International Publication 2012 / 130831, which is incorporated herein by reference in its entirety. International Publication 2012 / 130831 also describes methods for preparing such mutant Fc domains and determining their characterization, such as Fc receptor binding or effector function.

[0116] IgG4 antibodies exhibit reduced binding affinity to Fc receptors and decreased effector function compared to IgG1 antibodies. Therefore, in some embodiments, the target Fc domain of the targeted antibody is the IgG4Fc domain, particularly the human IgG4Fc domain. In one embodiment, the IgG4 target Fc domain includes an amino acid substitution at position S228, specifically the amino acid substitution S228P (numbered according to the Kabat EU index). To further reduce binding affinity to the Fc receptor and / or its effector function, in one embodiment, the IgG4 target Fc domain includes an amino acid substitution at position L235, specifically the amino acid substitution L235E (numbered according to the Kabat EU index). In another embodiment, the IgG4 target Fc domain includes an amino acid substitution at position P329, specifically the amino acid substitution P329G (numbered according to the Kabat EU index). In certain embodiments, the IgG4 target Fc domain includes amino acid substitutions at positions S228, L235, and P329, specifically including amino acid substitutions S228P, L235E, and P329G (numbering according to Kabat's EU index). Such IgG4Fc domain variants and their Fcγ receptor binding properties are described in PCT International Publication 2012 / 130831, which is incorporated herein by reference in whole.

[0117] In certain embodiments, the target Fc domains that exhibit reduced binding affinity to the Fc receptor and / or reduced effector function compared to the natural IgG1Fc domain are human IgG1Fc domains containing amino acid substitutions L234A, L235A and optionally P329G, or human IgG4Fc domains containing amino acid substitutions S228P, L235E and optionally P329G (numbering is according to the Kabat EU index).

[0118] In certain embodiments, N-glycosylation of the target Fc domain is removed. In one such embodiment, the target Fc domain includes an amino acid substitution at position N297, specifically an amino acid substitution replacing asparagine with alanine (N297A) or an amino acid substitution replacing aspartic acid with aspartic acid (N297D) (numbering according to Kabat's EU index).

[0119] In addition to the target Fc domains described herein, target Fc domains with reduced Fc receptor binding and / or effector function also include those having one or more substitutions at Fc domain residues 238, 265, 269, 270, 297, 327, and 329 (U.S. Patent No. 6,737,056) (numbering according to Kabat's EU index). Such target Fc variants include Fc variants having two or more substitutions at amino acid positions 265, 269, 270, 297, and 327, and include the so-called "DANA" Fc variant in which residues 265 and 297 are substituted with alanine (U.S. Patent No. 7,332,581).

[0120] The mutant target Fc domain can be prepared by deletion, substitution, insertion, or modification of amino acids using genetic or chemical methods well known in the art. Genetic methods may include site-directed mutagenesis of coding DNA sequences, PCR, gene synthesis, etc. Correct nucleotide changes can be confirmed, for example, by screening.

[0121] Binding to the Fc receptor can be readily determined, for example, by ELISA, or by surface plasmon resonance (SPR) using Fc receptors obtained by recombinant expression with standard equipment such as a BIAcore instrument (GE Healthcare). Appropriate such binding assays are described herein. Alternatively, the binding affinity of a target Fc domain or a targeted antibody containing a target Fc domain to the Fc receptor may be evaluated using cell lines known to express a specific Fc receptor (e.g., human NK cells expressing the FcγIIIa receptor).

[0122] The effector function of a target Fc domain or a targeted antibody containing such a target Fc domain can be measured by methods known in the art. Suitable assays for measuring ADCC are described herein. Other examples of in vitro assays for evaluating the ADCC activity of a molecule of interest are described in U.S. Patent No. 5,500,362, Hellstrom et al., Proc Natl Acad Sci USA 83,7059-7063 (1986) and Hellstrom et al., Proc Natl Acad Sci USA 82,1499-1502 (1985), U.S. Patent No. 5,821,337, Bruggemann et al., J Exp Med 166,1351-1361 (1987). Alternatively, non-radioactive assay methods may be used (e.g., ACTI® non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, CA), and CytoTox 96® non-radioactive cytotoxicity assay (Promega, Madison, Wisconsin)). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively or additionally, the ADCC activity of the molecule of interest may be evaluated in vivo in animal models, e.g., as disclosed in Clynes et al., Proc Natl Acad Sci USA 95, 652-656 (1998).

[0123] In some embodiments, the binding of the target Fc domain to complement components, particularly to C1q, is reduced. Therefore, in some embodiments where the target Fc domain is manipulated to have reduced effector function, this reduced effector function includes reduced CDC. A C1q binding assay may be performed to determine whether the targeted antibody can bind to C1q and thus possesses CDC activity. See, for example, the C1q and C3c binding ELISAs in International Publications 2006 / 029879 and 2005 / 100402. A CDC assay may also be performed to evaluate complement activation (see, for example, Gazzano-Santoro et al., J Immunol Methods 202, 163 (1996); Cragg et al., Blood 101, 1045-1052 (2003); and Cragg and Glennie, Blood 103, 2738-2743 (2004)).

[0124] At least one amino acid substitution in the second set At least one amino acid substitution of the second set is included in the half-life extension Fc domain of the immunoactivating Fc domain binding molecule, as illustrated in Figure 1. Thus, in one aspect of the present invention, the half-life extension Fc domain described herein includes at least one amino acid substitution of the second set. In one embodiment, at least one amino acid substitution of the second set includes at least one amino acid substitution that reduces the binding affinity and / or effector function of the half-life extension Fc domain to the Fc receptor. In one embodiment, the half-life extension Fc domain includes an amino acid substitution at a position selected from the group E233, L234, L235, N297, P331 and P329 (numbering according to Kabat's EU index). In a more specific embodiment, the half-life extension Fc domain includes an amino acid substitution at a position selected from the group L234, L235 and P329 (numbering according to Kabat's EU index). In some embodiments, the half-life extension Fc domain includes amino acid substitutions L234A and L235A (numbered according to Kabat's EU index). In one such embodiment, the half-life extension Fc domain is an IgG1Fc domain, particularly a human IgG1Fc domain. In one embodiment, the half-life extension Fc domain includes an amino acid substitution at position P329. In a more specific embodiment, the amino acid substitution is P329A or P329G, particularly P329G (numbered according to Kabat's EU index). In one embodiment, the half-life extension Fc domain includes an amino acid substitution at position P329 and includes further amino acid substitutions at positions selected from E233, L234, L235, N297 and P331 (numbered according to Kabat's EU index). In more specific embodiments, further amino acid substitutions include E233P, L234A, L235A, L235E, N297A, N297D, or P331S. In certain embodiments, the half-life extension Fc domain includes amino acid substitutions at positions P329, L234, and L235 (numbering according to Kabat's EU index).

[0125] In preferred embodiments, the half-life extension Fc domain includes amino acid substitutions L234A, L235A ("LALA", numbered according to the Kabat EU index). In more detailed embodiments, the half-life extension Fc domain includes amino acid substitutions L234A, L235A and P329G ("P329G LALA", numbered according to the Kabat EU index). In one such embodiment, the half-life extension Fc domain is an IgG1Fc domain, particularly a human IgG1Fc domain. The combination of amino acid substitutions "P329G LALA" almost completely eliminates the Fcγ receptor (and complement) binding of the human IgIgG1Fc domain, as described in International Publication No. 2012 / 130831, which is incorporated herein by reference in whole. International Publication No. 2012 / 130831 also describes methods for preparing such mutant Fc domains and determining their characteristics, such as Fc receptor binding or effector function.

[0126] In a preferred embodiment, the half-life extension Fc domain is IgG1, and at least one amino acid substitution in the second set includes a P329G substitution. In one such particular embodiment, the half-life extension Fc domain contains an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 29.

[0127] However, in some embodiments in which the Fc domain binding moiety can bind to a target Fc domain containing a P329G substitution, the half-life extension Fc domain preferably contains an amino acid substitution at position P329 (numbered according to the Kabat EU index) with an amino acid other than glycine (G). In one embodiment, at least one of the first set of amino acid substitutions described herein contains the amino acid substitution P329G (numbered according to the Kabat EU index), and at least one of the second set of amino acid substitutions contains a substitution at position P329 (numbered according to the Kabat EU index) with an amino acid other than glycine (G). In one embodiment, at least one of the second set of amino acid substitutions contains a substitution at position P329 (numbered according to the Kabat EU index) with an amino acid other than glycine (G), and such an amino acid cannot form a proline sandwich between the two conserved tryptophan side chains within the Fc gamma receptor, particularly within FcgRIIIa.

[0128] In such preferred embodiments, at least one amino acid substitution of the second set includes a substitution at position P329 (numbered according to the Kabat EU index) with an amino acid selected from the list consisting of arginine (R), leucine (L), isoleucine (I), and alanine (A). In more preferred embodiments, at least one amino acid substitution of the second set includes a substitution at position P329 (numbered according to the Kabat EU index) with arginine (R). The "LALA" amino acid substitutions, combined individually with the "P329R," "P329L," "P329I," and "P329A" amino acid substitutions, respectively, cause near-complete abolition of the Fcγ receptor (and complement) as described herein. In one embodiment, the immunoactivating Fc domain binding molecule includes a half-life extension Fc domain comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 29, SEQ ID NOs: 30, SEQ ID NOs: 31, SEQ ID NOs: 32, and SEQ ID NOs: 33.

[0129] In a preferred embodiment, the half-life extension Fc domain is IgG1, and at least one amino acid substitution in the second set includes a P329L substitution (numbered according to the Kabat EU index). In one such particular embodiment, the half-life extension Fc domain including the P329L substitution (numbered according to the Kabat EU index) includes an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 30.

[0130] In another preferred embodiment, the half-life extension Fc domain is IgG1, and at least one amino acid substitution in the second set includes a P329I substitution (numbered according to the Kabat EU index). In one such particular embodiment, the half-life extension Fc domain including the P329I substitution (numbered according to the Kabat EU index) includes an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 31.

[0131] In another preferred embodiment, the half-life extension Fc domain is IgG1, and at least one amino acid substitution in the second set includes a 329R substitution (numbered according to the Kabat EU index). In one such particular embodiment, the half-life extension Fc domain including the P329R substitution (numbered according to the Kabat EU index) includes an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 32.

[0132] In another preferred embodiment, the half-life extension Fc domain is IgG1, and at least one amino acid substitution in the second set includes a P329A substitution (numbered according to the Kabat EU index). In one such particular embodiment, the half-life extension Fc domain including the P329A substitution (numbered according to the Kabat EU index) includes an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 33.

[0133] IgG4 antibodies exhibit reduced binding affinity to Fc receptors and decreased effector function compared to IgG1 antibodies. Therefore, in some embodiments, the half-life extension Fc domain of the immunoactivating Fc domain binding molecule is the IgG4Fc domain, particularly the human IgG4Fc domain. In one embodiment, the IgG4 half-life extension Fc domain includes an amino acid substitution at position S228, specifically the amino acid substitution S228P (numbered according to the Kabat EU index). To further reduce binding affinity to the Fc receptor and / or its effector function, in one embodiment, the IgG4 half-life extension Fc domain includes an amino acid substitution at position L235, specifically the amino acid substitution L235E (numbered according to the Kabat EU index). In another embodiment, the IgG4 half-life extension Fc domain includes an amino acid substitution at position P329, specifically the amino acid substitution P329G (numbered according to the Kabat EU index). In certain embodiments, the IgG4 half-life extension Fc domain includes amino acid substitutions at positions S228, L235, and P329, specifically including amino acid substitutions S228P, L235E, and P329G (numbering according to Kabat's EU index). Such IgG4Fc domain variants and their Fcγ receptor binding properties are described in PCT International Publication 2012 / 130831, which is incorporated herein by reference in its entirety.

[0134] In certain embodiments, the half-life-extended Fc domains that exhibit reduced binding affinity to the Fc receptor and / or reduced effector function compared to the natural IgG1Fc domain are human IgG1Fc domains containing amino acid substitutions L234A, L235A and optionally P329G, or human IgG4Fc domains containing amino acid substitutions S228P, L235E and optionally P329G (numbering is according to the Kabat EU index).

[0135] In certain embodiments, the N-glycosylation of the half-life extension Fc domain is removed. In one such embodiment, the half-life extension Fc domain includes an amino acid substitution at position N297, specifically an amino acid substitution replacing asparagine with alanine (N297A) or an amino acid substitution replacing aspartic acid with aspartic acid (N297D) (numbering according to the Kabat EU index).

[0136] In addition to the half-life extension Fc domains described herein, half-life extension Fc domains with reduced Fc receptor binding and / or effector function also include those having one or more substitutions at Fc domain residues 238, 265, 269, 270, 297, 327, and 329 (U.S. Patent No. 6,737,056) (numbering according to Kabat's EU index). Such half-life extension Fc variants include Fc variants having two or more substitutions at amino acid positions 265, 269, 270, 297, and 327, and include the so-called "DANA" Fc variant in which residues 265 and 297 are substituted with alanine (U.S. Patent No. 7,332,581).

[0137] Mutant (substituted) half-life extension Fc domains can be prepared by amino acid deletion, substitution, insertion, or modification using genetic or chemical methods well known in the art. Genetic methods may include site-directed mutagenesis of coding DNA sequences, PCR, gene synthesis, etc. Correct nucleotide changes can be confirmed, for example, by screening.

[0138] Binding to the Fc receptor can be readily determined, for example, by ELISA, or by surface plasmon resonance (SPR) using Fc receptors obtained by recombinant expression with standard equipment such as the BIAcore instrument (GE Healthcare). Appropriate such binding assays are described herein. Alternatively, the binding affinity of an extended half-life Fc domain or an immunoactivating Fc domain-binding molecule containing an extended half-life Fc domain to the Fc receptor can be evaluated using cell lines known to express specific Fc receptors, such as human NK cells expressing the FcγIIIa receptor.

[0139] The effector function of an extended half-life Fc domain or an immunoactivating Fc domain-binding molecule containing such an extended half-life Fc domain can be measured by methods known in the art. Suitable assays for measuring ADCC are described herein. Other examples of in vitro assays for evaluating the ADCC activity of a molecule of interest are described in U.S. Patent No. 5,500,362, Hellstrom et al., Proc Natl Acad Sci USA 83,7059-7063 (1986) and Hellstrom et al., Proc Natl Acad Sci USA 82,1499-1502 (1985), U.S. Patent No. 5,821,337, Bruggemann et al., J Exp Med 166,1351-1361 (1987). Alternatively, non-radioactive assay methods may be used (e.g., ACTI® non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, CA), and CytoTox 96® non-radioactive cytotoxicity assay (Promega, Madison, Wisconsin)). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively or additionally, the ADCC activity of the molecule of interest may be evaluated in vivo in animal models, e.g., as disclosed in Clynes et al., Proc Natl Acad Sci USA 95, 652-656 (1998).

[0140] In some embodiments, the binding of the half-life extension Fc domain to complement components, particularly to C1q, is reduced. Therefore, in some embodiments where the half-life extension Fc domain is manipulated to have reduced effector function, this reduced effector function includes reduced CDC. A C1q binding assay may be performed to determine whether an immunoactivating Fc domain binding molecule can bind to C1q and therefore possesses CDC activity. See, for example, the C1q and C3c binding ELISAs in International Publication Nos. 2006 / 029879 and International Publication Nos. 2005 / 100402. A CDC assay may be performed to evaluate complement activation (see, for example, Gazzano-Santoro et al., J Immunol Methods 202, 163 (1996); Cragg et al., Blood 101, 1045-1052 (2003); and Cragg and Glennie, Blood 103, 2738-2743 (2004)).

[0141] Fc domain modification that promotes heterodimerization The immunoactivating Fc domain-binding molecule according to the present invention comprises different Fab molecules and immunoactivating moieties (e.g., Fab molecules, cytokines, ligands) fused to one or the other of two subunits of the half-life extension Fc domain, and thus the two subunits of the half-life extension Fc domain are typically contained in two non-identical polypeptide chains. Co-recombination expression of these polypeptides and subsequent dimerization result in several possible combinations of the two polypeptides. Therefore, to improve the yield and purity of the immunoactivating Fc domain-binding molecule in recombinant production, it would be advantageous to introduce modifications to the Fc domain of the immunoactivating Fc domain-binding molecule (i.e., the half-life extension Fc domain) that promote the association of the desired polypeptide.

[0142] Therefore, in certain embodiments, the half-life extension Fc domain according to the present invention includes modifications that facilitate the association of the first and second subunits of the Fc domain. The site of the longest protein-protein interaction between the two subunits of the human IgG Fc domain is located within the CH3 domain of the Fc domain. Therefore, in one embodiment, the modification is located within the CH3 domain of the Fc domain.

[0143] To enhance heterodimerization, several methods exist for modifying the CH3 domain of the Fc domain, which are well described, for example, in International Publications 96 / 27011, 98 / 050431, European Patent No. 1870459, 2007 / 110205, 2007 / 147901, 2009 / 089004, 2010 / 129304, 2011 / 90754, 2011 / 143545, 2012058768, 2013157954, and 2013096291. Typically, in all such methods, the CH3 domain of the first subunit of the Fc domain and the CH3 domain of the second subunit of the Fc domain are both manipulated in a complementary manner such that each CH3 domain (or the heavy chain containing it) can no longer homodimerize with itself, but heterodimerizes with the other complementaryly manipulated CH3 domain (the first and second CH3 domains heterodimerize, and no homodimer is formed between the two first or two second CH3 domains). These different methods for improved heavy chain heterodimerization are envisioned as different alternatives in combination with heavy-light chain modification in the immunoactivating Fc domain binding molecule of the present invention, which has reduced erroneous light chain pairing and Bence Jones-type byproducts (exchange / substitution of VH and VL in one binding arm and introduction of charged amino acids substituted with opposite charges at the CH1 / CL interface).

[0144] In a specific embodiment, the modification that facilitates the association of the first and second subunits of the half-life extension Fc domain is a so-called "knob-into-hole" modification, which includes a "knob" modification on one of the two subunits of the Fc domain and a "hole" modification on the other of the two subunits of the half-life extension Fc domain.

[0145] The knob-into-hole technique is described, for example, in U.S. Patents 5,731,168, 7,695,936, Ridgway et al., Prot Eng 9,617-621 (1996), and Carter, J Immunol Meth 248,7-15 (2001). Generally, this method involves introducing a bump ("knob") at the interface of a first polypeptide and a corresponding cavity ("hole") at the interface of a second polypeptide, so that the bump can be positioned within the cavity to promote heterodimerization and inhibit homodimerization. The bump is constructed by replacing a small amino acid side chain from the interface of the first polypeptide with a larger side chain (e.g., tyrosine or tryptophan). Complementary cavities of the same or similar size as the ridges are created at the contact surface of the second polypeptide by replacing larger amino acid side chains with smaller ones (e.g., alanine or threonine).

[0146] Therefore, in certain embodiments, in the CH3 domain of the first subunit of the half-life extension Fc domain, an amino acid residue is replaced with an amino acid residue having a larger side chain volume, thereby creating a bulge within the CH3 domain of the first subunit that is repositionable within the cavity in the CH3 domain of the second subunit, and in the CH3 domain of the second subunit of the half-life extension Fc domain, an amino acid residue is replaced with an amino acid residue having a smaller side chain volume, thereby creating a cavity within the CH3 domain of the second subunit, within which the bulge within the CH3 domain of the first subunit is repositionable.

[0147] Preferably, the amino acid residue having a larger side chain volume is selected from the group consisting of arginine (R), phenylalanine (F), tyrosine (Y), and tryptophan (W).

[0148] Preferably, the amino acid residue having a smaller side chain volume is selected from the group consisting of alanine (A), serine (S), threonine (T), and valine (V).

[0149] The ridges and cavities can be created by altering the nucleic acid encoding the polypeptide, for example, by site-directed mutagenesis or by peptide synthesis.

[0150] In a specific embodiment, in the first subunit ("knob" subunit) (CH3 domain) of the half-life extension Fc domain, the threonine residue at position 366 is replaced with a tryptophan residue (T366W), and in the second subunit (CH3 domain) of the half-life extension Fc domain ("whole" subunit), the tyrosine residue at position 407 is replaced with a valine residue (Y407V). In one embodiment, in the second subunit of the half-life extension Fc domain, the threonine residue at position 366 is further replaced with a serine residue (T366S), and the leucine residue at position 368 is replaced with an alanine residue (L368A) (numbering is according to the Kabat EU index).

[0151] In further embodiments, the serine residue at position 354 of the first subunit of the half-life extension Fc domain is further replaced with a cysteine ​​residue (S354C), or the glutamic acid residue at position 356 is replaced with a cysteine ​​residue (E356C), and the tyrosine residue at position 349 of the second subunit of the half-life extension Fc domain is further replaced with a cysteine ​​residue (Y349C) (numbering is based on Kabat's EU index). The introduction of these two cysteine ​​residues results in the formation of a disulfide bridge between the two subunits of the Fc domain, further stabilizing the dimer (Carter, J Immunol Methods 248, 7-15 (2001)).

[0152] In certain embodiments, the first subunit of the half-life extension Fc domain includes the amino acid substitutions S354C and T366W, and the second subunit of the half-life extension Fc domain includes the amino acid substitutions Y349C, T366S, L368A and Y407V (numbering according to Kabat's EU index).

[0153] In certain embodiments, the immunoactivating moiety is fused to the first subunit (including the "knob" modification) of the half-life extension Fc domain. While we do not wish to be constrained by theory, the fusion of the immunoactivating moiety with the knob-containing subunit of the half-life extension Fc domain minimizes the generation of an immunoactivating Fc domain-binding molecule containing (furthermore) two immunoactivating moieties (steric collision of the two knob-containing polypeptides).

[0154] Other techniques for CH3 modification that enhance heterodimerization have been considered as alternatives to the present invention and are described, for example, in International Publication Nos. 96 / 27011, 98 / 050431, European Patent No. 1870459, International Publication Nos. 2007 / 110205, 2007 / 147901, 2009 / 089004, 2010 / 129304, 2011 / 90754, 2011 / 143545, 2012 / 058768, 2013 / 157954, and 2013 / 096291.

[0155] In one embodiment, the heterodimerization method described in European Patent No. 1870459A1 is used instead. This method is based on the introduction of charged amino acids with opposite charges at specific amino acid positions at the CH3 / CH3 domain interface between two subunits of the half-life extension Fc domain. A preferred embodiment of the immunoactivating Fc domain binding molecule of the present invention is amino acid mutation R409D; K370E in one of the two CH3 domains (of the half-life extension Fc domain) and amino acid mutation D399K; E357K in the other CH3 domain of the half-life extension Fc domain (numbering according to the Kabat EU index).

[0156] In another embodiment, the immunoactivating Fc domain binding molecule of the present invention contains the amino acid mutation T366W in the CH3 domain of the first subunit of the half-life extension Fc domain, the amino acid mutations T366S, L368A, and Y407V in the CH3 domain of the second subunit of the half-life extension Fc domain, and further amino acid mutations R409D;K370E in the CH3 domain of the first subunit of the half-life extension Fc domain, and amino acid mutations D399K;E357K in the CH3 domain of the second subunit of the half-life extension Fc domain (numbering according to the Kabat EU index).

[0157] In another embodiment, the immunoactivating Fc domain binding molecule of the present invention contains amino acid mutations S354C, T366W in the CH3 domain of the first subunit of the half-life extension Fc domain, and amino acid mutations Y349C, T366S, L368A, Y407V in the CH3 domain of the second subunit of the half-life extension Fc domain, or the immunoactivating Fc domain binding molecule contains amino acid mutations in the CH3 domain of the first subunit of the half-life extension Fc domain. It contains variants Y349C and T366W, amino acid mutations S354C, T366S, L368A, and Y407V in the CH3 domain of the second subunit of the half-life-extended Fc domain, and further amino acid mutations R409D;K370E in the CH3 domain of the first subunit of the Fc domain, and amino acid mutations D399K;E357K in the CH3 domain of the second subunit of the Fc domain (all numbering is according to Kabat's EU index).

[0158] In one embodiment, the heterodimerization method described in International Publication No. 2013 / 157953 is used instead. In one embodiment, the first CH3 domain contains the amino acid mutation T366K, and the second CH3 domain contains the amino acid mutation L351D (numbering according to the Kabat EU index). In a further embodiment, the first CH3 domain contains a further amino acid mutation L351K. In a further embodiment, the second CH3 domain further contains amino acid mutations selected from Y349E, Y349D, and L368E (preferably L368E) (numbering according to the Kabat EU index).

[0159] In one embodiment, the heterodimerization method described in International Publication No. 2012 / 058768 is used instead. In one embodiment, the first CH3 domain contains amino acid mutations L351Y, Y407A, and the second CH3 domain contains amino acid mutations T366A, K409F. In further embodiments, the second CH3 domain contains further amino acid mutations at positions T411, D399, S400, F405, N390, or K392, e.g., (a) T411N, T411R, T411Q, T411K, T411D, T411E, or T411W, (b) D399R, D399W, D399Y, or D399K, (c) S400 (d) E, S400D, S400R or S400K, (e) F405I, F405M, F405T, F405S, F405V or F405W, (f) N390R, N390K or N390D, (f) K392V, K392M, K392R, K392L, K392F or K392E (numbering according to Kabat's EU index). In further embodiments, the first CH3 domain includes amino acid mutations L351Y and Y407A, and the second CH3 domain includes amino acid mutations T366V and K409F. In further embodiments, the first CH3 domain includes amino acid mutation Y407A, and the second CH3 domain includes amino acid mutations T366A and K409F. In further embodiments, the second CH3 domain further includes amino acid mutations K392E, T411E, D399R, and S400R (numbering according to the Kabat EU index).

[0160] In one embodiment, a heterodimerization technique described in International Publication No. 2011 / 143545 is used instead, having amino acid modifications at positions selected from the group consisting of, for example, 368 and 409 (numbering according to the Kabat EU index).

[0161] In one embodiment, the heterodimerization technique described in International Publication No. 2011 / 090762 is used instead, which also employs the technique of inserting a knob into the hole described above. In one embodiment, the first CH3 domain contains the amino acid mutation T366W, and the second CH3 domain contains the amino acid mutation Y407A. In another embodiment, the first CH3 domain contains the amino acid mutation T366Y, and the second CH3 domain contains the amino acid mutation Y407T (numbering is according to the Kabat EU index).

[0162] In one embodiment, the half-life extension Fc domain is of the IgG2 subclass, and the heterodimerization method described in International Publication No. 2010 / 129304 is used as an alternative.

[0163] In alternative embodiments, modifications that promote the association of the first and second subunits of the half-life extension Fc domain include modifications that intervene in the electrostatic maneuvering effect, as described, for example, in PCT International Publication 2009 / 089004. Generally, this method involves the substitution of one or more amino acid residues by charged amino acid residues at the contact surface of the two Fc domain subunits such that homodimerization is electrostatically undesirable, but heterodimerization is electrostatically desirable. In one such embodiment, the first CH3 domain includes an amino acid substitution with a negatively charged amino acid of K392 or N392 (e.g., glutamic acid (E) or aspartic acid (D), preferably K392D or N392D), and the second CH3 domain includes an amino acid substitution with a positively charged amino acid of D399, E356, D356 or E357 (e.g., lysine (K) or arginine (R), preferably D399K, E356K, D356K or E357K, more preferably D399K and E356K). In a further embodiment, the first CH3 domain further includes an amino acid substitution with a negatively charged amino acid of K409 or R409 (e.g., glutamic acid (E) or aspartic acid (D), preferably K409D or R409D). In further embodiments, the first CH3 domain further includes, or is replaced by, an amino acid substitution with a negatively charged amino acid at K439 and / or K370 (e.g., glutamic acid (E) or aspartic acid (D)) (all numbering according to Kabat's EU index).

[0164] In further embodiments, the heterodimerization method described in International Publication No. 2007 / 147901 is used instead. In one embodiment, the first CH3 domain contains amino acid mutations K253E, D282K, and K322D, and the second CH3 domain contains amino acid mutations D239K, E240K, and K292D (numbering is according to the Kabat EU index).

[0165] In yet another embodiment, the heterodimerization method described in International Publication No. 2007 / 110205 can be used instead.

[0166] In one embodiment, the first subunit of the Fc domain includes amino acid substitutions K392D and K409D, and the second subunit of the Fc domain includes amino acid substitutions D356K and D399K (numbering according to the Kabat EU index).

[0167] Fc domain binding portion The immune-activating Fc domain binding molecule of the present invention includes at least one Fc domain binding moiety that specifically binds to a target Fc domain, as illustrated in Figure 1.

[0168] Therefore, the immunoactivating Fc domain binding molecule of the present invention enables specific binding to the target Fc domain of targeted antibodies, i.e., therapeutic antibodies. As described herein, the present invention provides a versatile platform for directing specific effector functions to target cells. Targeted antibodies recognize and bind to target cells. The immunoactivating Fc domain binding molecule of the present invention recognizes and binds to the target Fc domain contained in the targeted antibody. The target Fc domain confers favorable pharmacokinetic properties and a favorable tissue-blood distribution ratio to the targeted antibody, i.e., therapeutic antibody, including a long serum half-life that contributes to good accumulation in target tissue. However, this can simultaneously lead to undesirable targeting of therapeutic antibodies to cells expressing Fc receptors rather than favorable antigen-carrying cells. Furthermore, co-activation of the Fc receptor signaling pathway can lead to cytokine release, resulting in excessive activation of cytokine receptors and serious side effects when therapeutic antibodies are systemically administered. Activation of immune cells other than T cells (that possess Fc receptors) may even reduce the effectiveness of therapeutic antibodies, as these immune cells may be destroyed. Therefore, therapeutic antibodies known in the field may be manipulated or mutated to exhibit reduced binding affinity to the Fc receptor and / or reduced effector function compared to, for example, the natural IgG1Fc domain.

[0169] In a preferred embodiment of the present invention, the targeted antibody is engineered or mutated to exhibit reduced binding affinity to the Fc receptor and / or reduced effector function. As described above herein, the target Fc domain may comprise at least one amino acid substitution of a first set. Thus, in a preferred embodiment, the targeted antibody has reduced binding affinity to the Fc receptor and / or reduced effector function. Simultaneously, at least one amino acid substitution of the first set is used to specifically target the target Fc domain via the Fc domain binding moiety. Fc domain binding moieties having desirable specificity are described below herein, as are methods for generating further Fc domain binding moieties having desirable specificity (e.g., immunization of the mammalian immune system with Fc domains comprising at least one amino acid substitution of a first set (see, for example, International Publication No. 2017 / 072210, incorporated herein by reference)).

[0170] In preferred embodiments, the Fc domain binding moiety does not specifically bind to the half-life extension Fc domain (to avoid crosslinking of two or more immunoactivating Fc domain binding molecules of the present invention). In such embodiments, it may be desirable to incorporate amino acid substitutions at the same amino acid positions in the target Fc domain and the half-life extension Fc domain. In one such embodiment, at least one amino acid substitution of the first set described herein reduces the binding affinity and / or effector function to the Fc receptor, and at least one amino acid substitution of the second set described herein includes one or more amino acid substitutions at the same amino acid positions as at least one amino acid substitution of the first set, wherein the amino acids in at least one amino acid substitution of the second set are substituted with different amino acids at the same positions compared to at least one amino acid substitution of the first set. In preferred embodiments, the Fc domain binding moiety does not bind to the half-life extension Fc domain. Such Fc domain binding moieties with desirable specificity can be generated, as described herein, for example, by immunizing the mammalian immune system with an Fc domain containing at least one amino acid substitution of a first set, and then screening for Fc domain binding moieties that do not specifically bind to an Fc domain containing at least one amino acid substitution of a second set, wherein at least one amino acid substitution of the first and / or second set increases binding to the Fc receptor and / or enhances effector function.

[0171] Exemplary Fc domain binding moieties that specifically bind to the target Fc domain (where at least one amino acid substitution in the first set includes a P329G substitution) rather than the half-life extension Fc domain (where at least one amino acid substitution in the second set does not include a P329G substitution, i.e., is wild-type at position P329 or includes a terminal amino acid substitution at position P329 other than glycine) are the anti-P329G(M-1.7.24)huIgG1 binder, comprising the CDR sequences of Sequence ID No. 1, 2, 3, 4, 5 and 6 (numbering according to Kabat's EU index), further described in International Publication No. 2017 / 072210. Another exemplary Fc domain binding moiety that specifically binds to the target Fc domain rather than the half-life extension Fc domain is the anti-AAA binder containing the CDR sequences SEQ ID NOs: 168, 169, 170, 171, 172, and 173 (numbering according to Kabat's EU index), further described in International Publication No. 2017 / 072210.

[0172] In a preferred embodiment of the present invention, an immunoactivating Fc domain binding molecule is provided, comprising an Fc domain binding moiety capable of specific binding to a mutant Fc domain containing the amino acid substitution P329G. The P329G mutation reduces binding to the Fcγ receptor and associated effector function. Therefore, the mutant Fc domain containing the P329G substitution binds to the Fcγ receptor with reduced or absent affinity compared to the unsubstituted Fc domain. In a preferred embodiment, the Fc domain binding moiety cannot bind to an Fc domain containing an amino acid substitution at position P329 (numbered according to the Kabat EU index) by an amino acid other than glycine (G). In one embodiment, the Fc domain binding moiety cannot bind to an Fc domain containing an amino acid substitution at position P329 (numbered according to the Kabat EU index) by an amino acid other than glycine (G), and such an amino acid cannot form a proline sandwich between the two conserved tryptophan side chains within the Fc gamma receptor, particularly within FcgRIIIa. In a preferred embodiment, the Fc domain binding portion can bind to an Fc domain containing the amino acid mutation P329G, but cannot bind to an Fc domain containing an amino acid substitution at position P329 by an amino acid selected from the list consisting of arginine (R), leucine (L), isoleucine (I), and alanine (A).

[0173] In certain embodiments, at least one amino acid substitution of the first set includes an amino acid substitution at position P329 (in IgG1 Fc). In preferred embodiments, at least one amino acid substitution of the first set includes an amino acid substitution P329G (numbered according to the Kabat EU index) in IgG1 Fc. In one embodiment, the Fc domain binding moiety is capable of specific binding to an IgG1 Fc domain containing the amino acid substitution P329G (numbered according to the Kabat EU index).

[0174] In one embodiment, the Fc domain binding moiety capable of specific binding to the IgG1 Fc domain, including the amino acid substitution P329G (numbering according to the Kabat EU index), (i) A heavy chain variable region (VH) comprising at least one heavy chain complementarity determining region selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 11, SEQ ID NO: 16 and SEQ ID NO: 21, (ii) A light chain variable region (VL) comprising at least one light chain complementarity determining region selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 26 comprising.

[0175] In one embodiment, the Fc domain binding portion capable of specific binding to an IgG1 Fc domain containing the amino acid substitution P329G (numbering according to Kabat's EU index) is (i) A heavy chain variable region (VH), wherein (a) The heavy chain complementarity determining region (CDR H)1 amino acid sequence of YSWIN (SEQ ID NO: 1), (b) A CDR H2 amino acid sequence selected from the group consisting of EITPDSSTINYTPSLKD (SEQ ID NO: 2), EITPDSSTINYTPSLKG (SEQ ID NO: 11) and EITPDSSTINYAPSLKG (SEQ ID NO: 16); and (c) The CDR H3 amino acid sequence PYDYGAWFAS (SEQ ID NO: 3) comprising a heavy chain variable region (VH), and (ii) A light chain variable region (VL), wherein (d) The light chain complementarity determining region (CDR L)1 amino acid sequence RSSTGAVTTSNYAN (SEQ ID NO: 4); (e) The CDR L2 amino acid sequence GTNKRAP (SEQ ID NO: 5); and (f) The CDR L3 amino acid sequence ALWYSNHWV (SEQ ID NO: 6) comprising a light chain variable region (VL).

[0176] In a particular embodiment, the Fc domain binding portion capable of specific binding to an IgG1 Fc domain containing the amino acid substitution P329G (numbering according to Kabat's EU index) is (i) A heavy chain variable region (VH), wherein (a) Heavy chain complementarity-determining region (CDR H)1 amino acid sequence RYWMN (SEQ ID NO: 1); (b) CDR H2 amino acid sequence EITPDSSTINYTPSLKD (SEQ ID NO: 2); and (c) CDR H3 amino acid sequence PYDYGAWFAS (SEQ ID NO: 3) A heavy chain variable region (VH) comprising (ii) A light chain variable region (VL) comprising (d) Light chain complementarity-determining region (CDR L)1 amino acid sequence RSSTGAVTTSNYAN (SEQ ID NO: 4); (e) CDR L2 amino acid sequence GTNKRAP (SEQ ID NO: 5); and (f) CDR L3 amino acid sequence ALWYSNHWV (SEQ ID NO: 6) A light chain variable region (VL) comprising

[0177] In a specific embodiment, the Fc domain binding portion capable of specific binding to an IgG1 Fc domain comprising the amino acid substitution P329G (numbering according to the Kabat EU index) is (i) A heavy chain variable region (VH) comprising (a) Heavy chain complementarity-determining region (CDR H)1 amino acid sequence RYWMN (SEQ ID NO: 1); (b) CDR H2 amino acid sequence EITPDSSTINYTPSLKG (SEQ ID NO: 11); and (c) CDR H3 amino acid sequence PYDYGAWFAS (SEQ ID NO: 3) A heavy chain variable region (VH) comprising (ii) A light chain variable region (VL) comprising (d) Light chain complementarity-determining region (CDR L)1 amino acid sequence RSSTGAVTTSNYAN (SEQ ID NO: 4); (e) CDR L2 amino acid sequence GTNKRAP (SEQ ID NO: 5); and (f) CDR L3 amino acid sequence ALWYSNHWV (SEQ ID NO: 6) A light chain variable region (VL) comprising

[0178] In one embodiment, the Fc domain binding moiety capable of specific binding to the IgG1 Fc domain, including the amino acid substitution P329G (numbering according to the Kabat EU index), (i) Heavy chain variable region (VH), (a) Heavy chain complementarity determining region (CDR H) 1 amino acid sequence RYWMN (SEQ ID NO: 1); (b) CDR H2 amino acid sequence EITPDSSTINYAPSLKG (SEQ ID NO: 16); and (c) CDR H3 amino acid sequence PYDYGAWFAS (SEQ ID NO: 3) The heavy chain variable region (VH), (ii) The light chain variable region (VL), (d) Light chain complementarity determination region (CDR L) 1 amino acid sequence RSSTGAVTTSNYAN (SEQ ID NO: 4); (e) CDR L2 amino acid sequence GTNKRAP (SEQ ID NO: 5); and (f) CDR L3 amino acid sequence ALWYSNHWV (SEQ ID NO: 6) It includes the light chain variable region (VL).

[0179] In one embodiment, the Fc domain binding portion capable of specific binding to the IgG1 Fc domain containing the amino acid substitution P329G (numbering according to Kabat's EU index) includes a heavy chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs. 7, SEQ ID NOs. 12, SEQ ID NOs. 17, and SEQ ID NOs. 19, and a light chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs. 8 and SEQ ID NOs. 13.

[0180] In one embodiment, the Fc domain binding moiety capable of specific binding to the IgG1 Fc domain, including the amino acid substitution P329G (numbering according to the Kabat EU index), (i) A heavy chain variable region sequence that is at least approximately 95%, 96%, 97%, 98%, 99%, or 100% identical to sequence number 7, and a light chain variable region sequence that is at least approximately 95%, 96%, 97%, 98%, 99%, or 100% identical to sequence number 8, (ii) A heavy chain variable region sequence that is at least approximately 95%, 96%, 97%, 98%, 99%, or 100% identical to sequence number 12, and a light chain variable region sequence that is at least approximately 95%, 96%, 97%, 98%, 99%, or 100% identical to sequence number 13, (iii) A heavy chain variable region sequence that is at least approximately 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 17, and a light chain variable region sequence that is at least approximately 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 13, or (iv) A heavy chain variable region sequence that is at least approximately 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 19, and a light chain variable region sequence that is at least approximately 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 13. Includes.

[0181] In one embodiment, the Fc domain binding portion capable of specific binding to the IgG1 Fc domain containing the amino acid substitution P329G (numbering according to Kabat's EU index) includes the heavy chain variable region sequence of SEQ ID NO: 7 and the light chain variable region of SEQ ID NO: 8.

[0182] In a preferred embodiment, the Fc domain binding portion capable of specific binding to the IgG1 Fc domain containing the amino acid substitution P329G (numbered according to the Kabat EU index) includes the heavy chain variable region sequence of SEQ ID NO: 12 and the light chain variable region of SEQ ID NO: 13.

[0183] In another preferred embodiment, the Fc domain binding portion capable of specific binding to the IgG1 Fc domain containing the amino acid substitution P329G (numbered according to the Kabat EU index) includes the heavy chain variable region sequence of SEQ ID NO: 17 and the light chain variable region of SEQ ID NO: 13.

[0184] In another preferred embodiment, the Fc domain binding portion capable of specific binding to the IgG1 Fc domain containing the amino acid substitution P329G (numbered according to the Kabat EU index) includes the heavy chain variable region sequence of SEQ ID NO: 19 and the light chain variable region of SEQ ID NO: 13.

[0185] In another embodiment, the Fc domain binding portion can bind to Fc domains containing amino acid substitutions I253A, H310A, and H435A (numbering according to Kabat's EU index). In one embodiment, the Fc domain binding portion cannot bind to Fc domains containing amino acid substitutions at positions I253, H310, and H435 (numbering according to Kabat's EU index) by amino acids other than alanine (A). In one embodiment, the Fc domain binding portion can bind to Fc domains containing amino acid substitutions I253A, H310A, and H435A, but cannot bind to Fc domains containing amino acid substitutions at positions I253, H310, and H435 (numbering according to Kabat's EU index) by amino acids other than alanine (A).

[0186] In one embodiment, at least one amino acid substitution of the first set includes amino acid substitutions located in IgG1 Fc, I253A, H310A, and H435A (numbered according to the Kabat EU index). In a preferred embodiment, at least one amino acid substitution of the first set includes amino acid substitutions I253A, H310A, and H435A (numbered according to the Kabat EU index) in IgG1 Fc. In one embodiment, the Fc domain binding portion is capable of specific binding to the IgG1 Fc domain containing amino acid substitutions I253A, H310A, and H435A (numbered according to the Kabat EU index).

[0187] In another embodiment, the Fc domain binding moiety capable of specific binding to an IgG1 Fc domain containing the amino acid mutations I253A, H310A and H435A (numbering according to the Kabat EU index) is (i) a heavy chain variable region (VH) comprising (a) a heavy chain complementarity determining region (CDR H)1 amino acid sequence SYGMS (SEQ ID NO: 168); (b) a CDR H2 amino acid sequence SSGGSY (SEQ ID NO: 169); and (c) a CDR H3 amino acid sequence LGMITTGYAMDY (SEQ ID NO: 170) and a heavy chain variable region (VH) comprising (ii) a light chain variable region (VL) comprising (d) a light chain complementarity determining region (CDR L)1 amino acid sequence RSSQTIVHSTGHTYLE (SEQ ID NO: 171); (e) a CDR L2 amino acid sequence KVSNRFS (SEQ ID NO: 172); and (f) a CDR L3 amino acid sequence ALWYSNHWV FQGSHVPYT (SEQ ID NO: 173) and a light chain variable region (VL) comprising.

[0188] In one embodiment, the Fc domain binding moiety capable of specific binding to an IgG1 Fc domain containing the amino acid mutations I253A, H310A and H435A (numbering according to the Kabat EU index) comprises a heavy chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 174 and a light chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 175.

[0189] In one embodiment, the Fc domain binding moiety capable of specific binding to an IgG1 Fc domain containing the amino acid mutations I253A, H310A and H435A (numbering according to the Kabat EU index) comprises the heavy chain variable region sequence of SEQ ID NO: 174 and the light chain variable of SEQ ID NO: 175. (i) a heavy chain variable region (VH) comprising

[0190] The present invention presents a bispecific immunoactivating Fc domain binding molecule. In a further embodiment, the present invention provides a bispecific immunoactivating Fc domain binding molecule, namely, an immunoactivating portion being an antigen-binding portion (e.g., a Fab molecule).

[0191] Therefore, the present invention relates to an immune-activating Fc domain binding molecule, (a) an Fc domain binding moiety that specifically binds to a target Fc domain comprising at least one amino acid substitution of the first set described herein, (b) an immune activation moiety which is a Fab molecule, scFv molecule, or scFab molecule, c) A half-life extension Fc domain consisting of first and second subunits capable of stable association Includes, This invention provides an immunoactivating Fc domain binding molecule in which the Fc domain binding portion does not specifically bind to the half-life extension Fc domain described herein.

[0192] The components of an immunoactivating fragment crystallizable (Fc) domain-binding molecule can be fused to each other in various configurations. Exemplary structures are shown in Figure 2. In some embodiments, the immunoactivating portion is a Fab molecule fused at the C-terminus of the Fab heavy chain to the N-terminus of a first or second subunit of the half-life extension Fc domain. In one such embodiment, the Fc domain-binding portion is a Fab molecule fused at the C-terminus of the Fab heavy chain to the N-terminus of the immunoactivating portion, which is a second Fab molecule. In such specific embodiments, the immunoactivating Fc domain-binding molecule essentially consists of first and second Fab molecules, a half-life extension Fc domain composed of first and second subunits, and optionally one or more peptide linkers, where the first Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second Fab molecule, and the second Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of a first or second subunit of the half-life extension Fc domain. Such configurations are schematically shown in Figures 2G and 2K. Optionally, the Fab light chains of the first Fab molecule and the Fab light chains of the second Fab molecule may be additionally fused to each other.

[0193] In another specific embodiment, the immunoactivating Fc domain binding molecule essentially consists of an Fc domain binding portion, which is a Fab molecule, and an immunoactivating portion, which is a second Fab molecule, and the half-life extension Fc domain consists of first and second subunits and optionally one or more peptide linkers, with the first and second Fab molecules each fused to the N-terminus of one of the Fc domain subunits at the C-terminus of the Fab heavy chain. Such configurations are schematically shown in Figures 2A and 2D. The first and second Fab molecules may be fused to the half-life extension Fc domain directly or via peptide linkers. In a particular embodiment, the first and second Fab molecules are fused to the Fc domain by an immunoglobulin hinge region, respectively. In a particular embodiment, the immunoglobulin hinge region is a human IgG1 hinge region, and in particular, the Fc domain is an IgG1Fc domain.

[0194] In other embodiments, the Fc domain binding portion is a Fab molecule fused to the N-terminus of a first or second subunit of an extended half-life Fc domain at the C-terminus of the Fab heavy chain. In one such embodiment, the immunoactivating portion is a second Fab molecule fused to the N-terminus of the Fab heavy chain of a first Fab molecule at the C-terminus of the Fab heavy chain. In such specific embodiments, the immunoactivating Fc domain binding molecule essentially consists of first and second Fab molecules, an Fc domain composed of first and second subunits, and optionally one or more peptide linkers, where the second Fab molecule is fused to the N-terminus of the Fab heavy chain of the first Fab molecule at the C-terminus of the Fab heavy chain, and the first Fab molecule is fused to the N-terminus of a first or second subunit of an Fc domain at the C-terminus of the Fab heavy chain. Such configurations are schematically shown in Figures 2H and 2L. Optionally, the Fab light chains of the first Fab molecule and the Fab light chains of the second Fab molecule may be additionally fused to each other.

[0195] Multiple Fab molecules can be fused to each other, directly, or via peptide linkers containing one or more amino acids, typically about 2 to 20 amino acids, to their half-life extension Fc domains. Peptide linkers are known in the art and are described herein. Suitable non-immunogenic peptide linkers include, for example, (G4S) n (SG4) n (G4S) n Or G4 (SG4) n Peptide linkers are mentioned. "n" is generally an integer from 1 to 10, typically from 2 to 4. In one embodiment, the peptide linker has a length of at least 5 amino acids, in one embodiment it has a length of 5 to 100 amino acids, and in a further embodiment it has a length of 10 to 50 amino acids. In one embodiment the linker peptide is (GxS) n or (GxS) n G mThe peptide linker is (G4S)2, where G=glycine, S=serine, and (x=3, n=3, 4, 5 or 6, m=0, 1, 2 or 3), or (x=4, n=2, 3, 4 or 5, m=0, 1, 2 or 3), where in one embodiment x=4, n=2 or 3, and in a further embodiment x=4, n=2. In one embodiment, the peptide linker is (G4S)2. The peptide linker particularly suitable for fusing the Fab light chains of the first and second Fab molecules to each other is (G4S)2. An exemplary peptide linker suitable for linking to the Fab heavy chains of the first and second Fab fragments includes the sequence (D)-(G4S)2). Another exemplary peptide linker suitable for linking to the Fab heavy chains of the first and second Fab fragments includes the sequence (G4SG5). In addition, the linker may include (part of) an immunoglobulin hinge region. In particular, when the Fab molecule fuses to the N-terminus of the Fc domain subunit, it may fuse with or without a further peptide linker via the immunoglobulin hinge region or a portion thereof.

[0196] In some cases, for example, to optimize targeting to a target Fc domain or to enable crosslinking of the target molecule, it may be advantageous to have an immunoactivating Fc domain binding molecule containing two or more Fc domain binding moieties as described herein (see examples shown in 2B, 2C, 2E, 2F, 2I, 2J, 2M, or 2N).

[0197] Accordingly, in certain embodiments, the immunoactivating Fc domain binding molecule of the present invention further comprises a third Fab molecule that specifically binds to a target Fc domain comprising at least one amino acid substitution of the first set described herein. In one embodiment, the third Fab molecule is a conventional Fab molecule. In one embodiment, the third Fab molecule is identical to the first Fab molecule (i.e., the first and third Fab molecules comprise the same heavy-chain and light-chain amino acid sequences and have the same domain configuration (i.e., conventional or crossover)). In certain embodiments, the second Fab molecule specifically binds to an immunoactivating antigen, particularly CD3, and the first and third Fab molecules specifically bind to a target Fc domain comprising at least one amino acid substitution of the first set described herein.

[0198] In alternative embodiments, the immunoactivating Fc domain binding molecule of the present invention further comprises a third Fab molecule that specifically binds to an immunoactivating antigen, particularly CD3. In such an embodiment, the third Fab molecule is a crossover Fab molecule (a Fab molecule in which the variable domains VH and VL of the Fab heavy and light chains are exchanged / substituted with each other). In such an embodiment, the third Fab molecule is identical to the second Fab molecule (i.e., the second and third Fab molecules contain the same heavy and light chain amino acid sequences and have the same domain configuration (i.e., conventional or crossover)). In such an embodiment, the first Fab molecule specifically binds to an immunoactivating antigen, particularly CD3, and the second and third Fab molecules specifically bind to a target Fc domain containing at least one amino acid substitution of the first set described herein.

[0199] In one embodiment, the third Fab molecule is fused to the N-terminus of the first or second subunit of the Fc domain at the C-terminus of the Fab heavy chain.

[0200] In a particular embodiment, the second and third Fab molecules are each fused to the N-terminus of one subunit of the Fc domain at the C-terminus of the Fab heavy chain, and the first Fab molecule is fused to the N-terminus of the Fab heavy chain of the second Fab molecule at the C-terminus of the Fab heavy chain. In one such specific embodiment, the immunoactivating Fc domain binding molecule essentially consists of first, second and third Fab molecules, a half-life extension Fc domain composed of first and second subunits, and optionally one or more peptide linkers, wherein the first Fab molecule is fused to the N-terminus of the Fab heavy chain of the second Fab molecule at the C-terminus of the Fab heavy chain, the second Fab molecule is fused to the N-terminus of the first subunit of the half-life extension Fc domain at the C-terminus of the Fab heavy chain, and the third Fab molecule is fused to the N-terminus of the second subunit of the half-life extension Fc domain at the C-terminus of the Fab heavy chain. Such configurations are schematically shown in Figures 2B and 2E (a specific embodiment in which the third Fab molecule is a conventional Fab molecule, preferably identical to the first Fab molecule), and Figures 2I and 2M (an alternative embodiment in which the third Fab molecule is a crossover Fab molecule, preferably identical to the second Fab molecule). The second and third Fab molecules may be fused directly or via a peptide linker to the half-life extension Fc domain. In a specific embodiment, the second and third Fab molecules are fused to the half-life extension Fc domain, respectively, by an immunoglobulin hinge region. In a specific embodiment, the immunoglobulin hinge region is a human IgG1 hinge region, and in particular, the half-life extension Fc domain is an IgG1Fc domain. Optionally, the Fab light chains of the first Fab molecule and the second Fab molecule may be additionally fused to each other.

[0201] In another embodiment, the first and third Fab molecules are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the half-life extension Fc domain, and the second Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first Fab molecule. In one such specific embodiment, the immunoactivating Fc domain binding molecule essentially consists of first, second and third Fab molecules, a half-life extension Fc domain composed of the first and second subunits, and optionally one or more peptide linkers, wherein the second Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first Fab molecule, the first Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit of the Fc domain, and the third Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the second subunit of the Fc domain. Such configurations are schematically shown in Figures 2C and 2F (a specific embodiment in which the third Fab molecule is a conventional Fab molecule, preferably identical to the first Fab molecule), and Figures 2J and 2N (an alternative embodiment in which the third Fab molecule is a crossover Fab molecule, preferably identical to the second Fab molecule). The first and third Fab molecules may be fused directly or via a peptide linker to the half-life extension Fc domain. In a specific embodiment, the first and third Fab molecules are fused to the half-life extension Fc domain, respectively, by an immunoglobulin hinge region. In a specific embodiment, the immunoglobulin hinge region is a human IgG1 hinge region, and in particular, the Fc domain is an IgG1Fc domain. Optionally, the Fab light chains of the first Fab molecule and the Fab light chains of the second Fab molecule may be additionally fused to each other.

[0202] In the configuration of an immunoactivated Fc domain-binding molecule in which the Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of each subunit of the half-life extension Fc domain via an immunoglobulin hinge region, the two Fab molecules, the hinge region, and the half-life extension Fc domain essentially form an immunoglobulin molecule. In certain embodiments, the immunoglobulin molecule is an IgG class immunoglobulin. In even more specific embodiments, the immunoglobulin is an IgG1 subclass immunoglobulin. In another embodiment, the immunoglobulin is an IgG4 subclass immunoglobulin. In yet another embodiment, the immunoglobulin is a human immunoglobulin. In yet another embodiment, the immunoglobulin is a chimeric immunoglobulin or a humanized immunoglobulin.

[0203] In some of the immunoactivating Fc domain binding molecules of the present invention, the Fab light chain of the first Fab molecule and the Fab light chain of the second Fab molecule are optionally fused to each other via a peptide linker. Depending on the structures of the first and second Fab molecules, the Fab light chain of the first Fab molecule may be fused at its C-terminus to the N-terminus of the Fab light chain of the second Fab molecule, or the Fab light chain of the second Fab molecule may be fused at its C-terminus to the N-terminus of the Fab light chain of the first Fab molecule. The fusion of the Fab light chains of the first and second Fab molecules further reduces mispairing of incompatible Fab heavy and light chains and also reduces the number of plasmids required for the expression of some of the immunoactivating Fc domain binding molecules of the present invention.

[0204] In certain embodiments, the immunoactivating Fc domain binding molecule according to the present invention has a carboxy-terminal peptide bond between the Fab light chain variable region of the second Fab molecule and the Fab heavy chain constant region of the second Fab molecule (i.e., the second Fab molecule includes a crossover Fab heavy chain, and the heavy chain variable region replaces the light chain variable region), and the Fab heavy chain constant region of the second Fab molecule is an Fc domain subunit (VL (2) -CH1 (2)-CH2-CH3(-CH4)) and polypeptides sharing a carboxy-terminal peptide bond, and the Fab heavy chain of the first Fab molecule, form an Fc domain subunit (VH (1) -CH1 (1) The molecule contains a polypeptide sharing a carboxy-terminal peptide bond with a -CH2-CH3(-CH4)) molecule. In some embodiments, the immunoactivating Fc domain-binding molecule further comprises a polypeptide, and the Fab heavy chain variable region of the second Fab molecule is the Fab light chain constant region (VH) of the second Fab molecule. (2) -CL (2) ) and the Fab light chain polypeptide (VL) of the first Fab molecule (1) -CL (1) ) and share a carboxy-terminal peptide bond. In certain embodiments, the polypeptides are covalently bonded, for example, by disulfide bonds.

[0205] In some embodiments, the immunoactivating Fc domain binding molecule is such that the Fab light chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the second Fab molecule (i.e., the second Fab molecule includes a crossover Fab heavy chain, and the heavy chain variable region is replaced by the light chain variable region), the Fab heavy chain constant region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain of the first Fab molecule, and the Fab heavy chain of the first Fab molecule shares a carboxy-terminal peptide bond with the Fc domain subunit (VL polypeptide). (2) -CH1 (2) -VH (1) -CH1 (1)In other embodiments, the immunoactivating Fc domain includes a polypeptide (VH) in which the Fab heavy chain of the first Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain variable region of the second Fab molecule, and the Fab light chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the second Fab molecule (i.e., the second Fab molecule includes a crossover Fab heavy chain, in which the heavy chain variable region is replaced by the light chain variable region), and the Fab heavy chain constant region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fc domain subunit. (1) -CH1 (1) -VL (2) -CH1 (2) Includes -CH2-CH3(-CH4)).

[0206] In some of these embodiments, the immune-activating Fc domain binding molecule is a crossover Fab light chain polypeptide (VH) of the second Fab molecule, in which the Fab heavy chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the second Fab molecule. (2) -CL (2) ) and the Fab light chain polypeptide (VL) of the first Fab molecule (1) -CL (1) ) further comprises. In some other embodiments of these embodiments, where appropriate, the immunoactivating Fc domain binding molecule comprises a polypeptide (VL) in which the Fab light chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the second Fab molecule, and the Fab heavy chain constant region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain polypeptide of the first Fab molecule. (2) -CH1 (2) -VL (1) -CL (1) ), or a polypeptide (VL) in which the Fab light chain polypeptide of the first Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain variable region of the second Fab molecule, and the Fab heavy chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the second Fab molecule. (1) -CL (1)-VH (2) -CL (2) ) further includes.

[0207] The immune-activating Fc domain binding molecules in these embodiments are (i) an Fc domain subunit polypeptide (CH2-CH3(-CH4)), or (ii) a polypeptide in which the Fab heavy chain of the third Fab molecule shares a carboxy-terminal peptide bond with the Fc domain subunit (VH (3) -CH1 (3) -CH2-CH3(-CH4)) and the Fab light chain polypeptide (VL) of the third Fab molecule (3) -CL (3) ) may further include. In certain embodiments, the polypeptides are covalently bonded, for example, by disulfide bonds.

[0208] In some embodiments, for example, when a short half-life of the immune-activating Fc domain binding molecule is preferred, the immune-activating Fc domain binding molecule does not contain an Fc domain. Therefore, the present invention provides an immune-activating Fc domain binding molecule lacking an Fc domain (see Figures 2O-2Z for exemplary formats). In some embodiments, a first Fab molecule is fused to the N-terminus of the Fab heavy chain of a second Fab molecule at the C-terminus of the Fab heavy chain. In certain such embodiments, the immune-activating Fc domain binding molecule does not contain an Fc domain. In certain embodiments, the immune-activating Fc domain binding molecule essentially consists of first and second Fab molecules and optionally one or more peptide linkers, where the first Fab molecule is fused to the N-terminus of the Fab heavy chain of a second Fab molecule at the C-terminus of the Fab heavy chain. Such configurations are schematically shown in Figures 2O and 2S. In other embodiments, the second Fab molecule is fused to the N-terminus of the Fab heavy chain of a first Fab molecule at the C-terminus of the Fab heavy chain. In certain such embodiments, the immunoactivating Fc domain binding molecule does not contain an Fc domain. In certain embodiments, the immunoactivating Fc domain binding molecule essentially consists of first and second Fab molecules and optionally one or more peptide linkers, wherein the second Fab molecule is fused to the N-terminus of the Fab heavy chain of the first Fab molecule at the C-terminus of the Fab heavy chain. Such configurations are schematically shown in Figures 2P and 2T. In some embodiments, the first Fab molecule is fused to the N-terminus of the Fab heavy chain of the second Fab molecule at the C-terminus of the Fab heavy chain, and the immunoactivating Fc domain binding molecule further comprises a third Fab molecule, wherein the third Fab molecule is fused to the N-terminus of the Fab heavy chain of the first Fab molecule at the C-terminus of the Fab heavy chain. In such certain embodiments, the third Fab molecule is a conventional Fab molecule. In other such embodiments, the third Fab molecule is a crossover Fab molecule as described herein, i.e., a Fab molecule in which the variable domains VH and VL of the Fab heavy chain and Fab light chain are exchanged / substituted for each other.In certain such embodiments, the immunoactivating Fc domain binding molecule essentially consists of first, second, and third Fab molecules, and optionally one or more peptide linkers, wherein the first Fab molecule is fused to the N-terminus of the Fab heavy chain of the second Fab molecule at the C-terminus of the Fab heavy chain, and the third Fab molecule is fused to the N-terminus of the Fab heavy chain of the first Fab molecule at the C-terminus of the Fab heavy chain. Such configurations are schematically shown in Figures 2Q and 2U (a particular embodiment in which the third Fab molecule is a conventional Fab molecule, preferably identical to the first Fab molecule). In some embodiments, the first Fab molecule is fused to the N-terminus of the Fab heavy chain of the second Fab molecule at the C-terminus of the Fab heavy chain, and the immunoactivating Fc domain binding molecule further comprises a third Fab molecule, wherein the third Fab molecule is fused to the C-terminus of the Fab heavy chain of the second Fab molecule at the N-terminus of the Fab heavy chain. In certain such embodiments, the third Fab molecule is a crossover Fab molecule as described herein, i.e., a Fab molecule in which the variable domains VH and VL of the Fab heavy chain and Fab light chain are exchanged / substituted with each other. In other such embodiments, the third Fab molecule is a conventional Fab molecule. In certain such embodiments, the immunoactivating Fc domain binding molecule essentially consists of first, second and third Fab molecules and optionally one or more peptide linkers, wherein the first Fab molecule is fused to the N-terminus of the Fab heavy chain of the second Fab molecule at the C-terminus of the Fab heavy chain, and the third Fab molecule is fused to the C-terminus of the Fab heavy chain of the second Fab molecule at the N-terminus of the Fab heavy chain. Such configurations are schematically shown in Figures 2W and 2Y (a particular embodiment in which the third Fab molecule is a crossover Fab molecule, preferably identical to the second Fab molecule). In some embodiments, a second Fab molecule is fused to the N-terminus of the Fab heavy chain of a first Fab molecule at the C-terminus of the Fab heavy chain, and the immunoactivating Fc domain binding molecule further comprises a third Fab molecule, the third Fab molecule being fused to the C-terminus of the Fab heavy chain of a first Fab molecule at the N-terminus of the Fab heavy chain. In such specific embodiments, the third Fab molecule is a conventional Fab molecule.In other such embodiments, the third Fab molecule is a crossover Fab molecule as described herein, i.e., a Fab molecule in which the variable domains VH and VL of the Fab heavy chain and Fab light chain are exchanged / substituted with each other. In certain such embodiments, the immunoactivating Fc domain binding molecule essentially consists of the first, second and third Fab molecules and optionally one or more peptide linkers, wherein the second Fab molecule is fused to the N-terminus of the Fab heavy chain of the first Fab molecule at the C-terminus of the Fab heavy chain, and the third Fab molecule is fused to the C-terminus of the Fab heavy chain of the first Fab molecule at the N-terminus of the Fab heavy chain. Such configurations are schematically shown in Figures 2R and 2V (a particular embodiment in which the third Fab molecule is a conventional Fab molecule, preferably identical to the first Fab molecule). In some embodiments, a second Fab molecule is fused to the N-terminus of the Fab heavy chain of a first Fab molecule at the C-terminus of the Fab heavy chain, and the immunoactivating Fc domain binding molecule further comprises a third Fab molecule, the third Fab molecule being fused to the N-terminus of the Fab heavy chain of a second Fab molecule at the C-terminus of the Fab heavy chain. In certain such embodiments, the third Fab molecule is a crossover Fab molecule as described herein, i.e., a Fab molecule in which the variable domains VH and VL of the Fab heavy chain and Fab light chain are exchanged / substituted with each other. In other such embodiments, the third Fab molecule is a conventional Fab molecule. In certain such embodiments, the immunoactivating Fc domain binding molecule essentially consists of first, second and third Fab molecules and optionally one or more peptide linkers, the second Fab molecule being fused to the N-terminus of the Fab heavy chain of a first Fab molecule at the C-terminus of the Fab heavy chain, and the third Fab molecule being fused to the N-terminus of the Fab heavy chain of a second Fab molecule at the C-terminus of the Fab heavy chain. Such a configuration is schematically shown in Figures 2X and 2Z (a specific embodiment in which the third Fab molecule is a crossover Fab molecule, preferably identical to the first Fab molecule).

[0209] In certain embodiments, the immunoactivating Fc domain binding molecule according to the present invention comprises a polypeptide (VH) in which the Fab heavy chain of the first Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain variable region of the second Fab molecule, and the Fab light chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the second Fab molecule (i.e., the second Fab molecule contains a crossover Fab heavy chain, and the heavy chain variable region is replaced by the light chain variable region). (1) -CH1 (1) -VL (2) -CH1 (2) ) includes. In some embodiments, the immunoactivating Fc domain binding molecule further comprises a polypeptide, and the Fab heavy chain variable region of the second Fab molecule comprises the Fab light chain constant region (VH) of the second Fab molecule. (2) -CL (2) ) and the Fab light chain polypeptide (VL) of the first Fab molecule (1) -CL (1) ) and share a carboxy-terminal peptide bond.

[0210] In certain embodiments, the immunoactivating Fc domain binding molecule according to the present invention has a Fab light chain variable region of a second Fab molecule that shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the second Fab molecule (i.e., the second Fab molecule contains a crossover Fab heavy chain, and the heavy chain variable region is replaced by the light chain variable region), and the Fab heavy chain constant region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain of the first Fab molecule (VL (2) -CH1 (2) -VH (1) -CH1 (1) ) includes. In some embodiments, the immunoactivating Fc domain binding molecule further comprises a polypeptide, and the Fab heavy chain variable region of the second Fab molecule comprises the Fab light chain constant region (VH) of the second Fab molecule. (2) -CL (2) ) and the Fab light chain polypeptide (VL) of the first Fab molecule (1) -CL (1) ) and share a carboxy-terminal peptide bond.

[0211] In certain embodiments, the immunoactivating Fc domain binding molecule according to the present invention comprises a polypeptide (VH) in which the Fab heavy chain of a third Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain of a first Fab molecule, the Fab heavy chain of the first Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain variable region of a second Fab molecule, and the Fab light chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the second Fab molecule (i.e., the second Fab molecule contains a crossover Fab heavy chain, and the heavy chain variable region is replaced by the light chain variable region). (3) -CH1 (3) -VH (1) -CH1 (1) -VL (2) -CH1 (2) ) includes. In some embodiments, the immunoactivating Fc domain binding molecule further comprises a polypeptide, and the Fab heavy chain variable region of the second Fab molecule comprises the Fab light chain constant region (VH) of the second Fab molecule. (2) -CL (2) ) and the Fab light chain polypeptide (VL) of the first Fab molecule (1) -CL (1) ) and share a carboxy-terminal peptide bond. In some embodiments, the immunoactivating Fc domain-binding molecule is the Fab light chain polypeptide (VL) of the third Fab molecule. (3) -CL (3) ) further includes.

[0212] In certain embodiments, the immunoactivating Fc domain binding molecule according to the present invention comprises a polypeptide (VL) in which the Fab light chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the second Fab molecule (i.e., the second Fab molecule includes a crossover Fab heavy chain, and the heavy chain variable region is replaced by the light chain variable region), the Fab heavy chain constant region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain of the first Fab molecule, and the Fab heavy chain of the first Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain of the third Fab molecule. (2) -CH1 (2)-VH (1) -CH1 (1) -VH (3) -CH1 (3) ) includes. In some embodiments, the immunostimulatory Fc domain-binding molecule further includes a polypeptide, and the Fab heavy chain variable region of the second Fab molecule is the Fab light chain constant region of the second Fab molecule (VH (2) -CL (2) ) and the Fab light chain polypeptide of the first Fab molecule (VL (1) -CL (1) ) share a carboxy-terminal peptide bond. In some embodiments, the immunostimulatory Fc domain-binding molecule further includes the Fab light chain polypeptide of the third Fab molecule (VL (3) -CL (3) ).

[0213] In certain embodiments, the immunostimulatory Fc domain-binding molecule according to the present invention is such that the Fab heavy chain of the first Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain variable region of the second Fab molecule and then shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the second Fab molecule (i.e., the second Fab molecule includes a crossover Fab heavy chain and the heavy chain variable region is replaced by the light chain variable region), and the Fab heavy chain constant region of the first Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain variable region of the third Fab molecule and then shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the third Fab molecule (i.e., the third Fab molecule includes a crossover Fab heavy chain and the heavy chain variable region is replaced by the light chain variable region) polypeptide (VH (2) -CH1 (2) -VL (1) -CH1 (1) -VL (3) -CH1 (3) ). In some embodiments, the immunostimulatory Fc domain-binding molecule further includes a polypeptide, and the Fab heavy chain variable region of the second Fab molecule is the Fab light chain constant region of the second Fab molecule (VH (2) -CL (2) ) and the Fab light chain polypeptide of the first Fab molecule (VL (1) -CL(1) ) and share a carboxy-terminal peptide bond. In some embodiments, the immunoactivating Fc domain binding molecule has a polypeptide (VH) in which the Fab heavy chain variable region of the third Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the third Fab molecule. (3) -CL (3) ) further includes.

[0214] In certain embodiments, the immunoactivating Fc domain binding molecule according to the present invention has a carboxy-terminal peptide bond shared between the Fab light chain variable region of the third Fab molecule and the Fab heavy chain constant region of the third Fab molecule (i.e., the third Fab molecule includes a crossover Fab heavy chain, with the heavy chain variable region replaced by the light chain variable region), the Fab heavy chain constant region of the third Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain variable region of the second Fab molecule, the Fab light chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the second Fab molecule (i.e., the second Fab molecule includes a crossover Fab heavy chain, with the heavy chain variable region replaced by the light chain variable region), and the Fab heavy chain constant region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain of the first Fab molecule (VL (3) -CH1 (3) -VL (2) -CH1 (2) -VH (1) -CH1 (1) ) comprises polypeptide. In some embodiments, the immunoactivating Fc domain binding molecule further comprises polypeptide, and the Fab heavy chain variable region of the second Fab molecule comprises the Fab light chain constant region (VH) of the second Fab molecule. (2) -CL (2) ) and the Fab light chain polypeptide (VL) of the first Fab molecule (1) -CL (1) ) and share a carboxy-terminal peptide bond. In some embodiments, the immunoactivating Fc domain binding molecule has a polypeptide (VH) in which the Fab heavy chain variable region of the third Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the third Fab molecule. (3) -CL (3)) further includes.

[0215] According to any of the embodiments described above, the components of the immunoactivating Fc domain binding molecule (e.g., Fab molecule, Fc domain) may be fused directly or via various linkers, particularly via peptide linkers containing one or more amino acids, typically about 2 to 20 amino acids, as described herein or known in the art. Suitable non-immunogenic peptide linkers include, for example, (G4S) n (SG4) n (G4S) n Or G4 (SG4) n It contains a peptide linker, where "n" is usually an integer between 1 and 10, typically between 2 and 4. charge modification

[0216] In some embodiments, the immunoactivating Fc domain binding molecule of the present invention is bispecific, i.e., it comprises at least two antigen-binding moieties capable of specific binding to two different antigenic determinants. In some embodiments, the antigen-binding moiety is a Fab molecule (i.e., an antigen-binding domain composed of a heavy chain and a light chain, each containing a variable domain and a constant domain). In one embodiment, the Fab molecule is human. In another embodiment, the Fab molecule is humanized. In yet another embodiment, the Fab molecule comprises human heavy chain and light chain constant domains.

[0217] At least one of the antigen-binding moieties is a crossover Fab molecule. Such modifications reduce heavy-light chain mismatches from different Fab molecules, thereby increasing the yield and purity of the immunoactivating Fc domain-binding molecule of the present invention in recombinant production. In certain crossover Fab molecules useful for the immunoactivating Fc domain-binding molecule of the present invention, the variable domains of the Fab light chain and Fab heavy chain (VL and VH, respectively) are replaced. However, even with this domain exchange, the preparation of the immunoactivating Fc domain-binding molecule may include certain byproducts due to so-called Bence Jones-type interactions between poorly paired heavy and light chains (see Schaefer et al, PNAS, 108(2011)11187-11191). To further reduce mispairing of heavy and light chains from different Fab molecules and thus increase the purity and yield of the desired immunoactivating Fc domain binding molecule, according to the present invention, charged amino acids with opposite charges are introduced at specific amino acid positions in the CH1 domain and CL domain of either a Fab molecule that specifically binds to a target cell antigen or a Fab molecule(s) that specifically binds to an immunoactivating antigen. The charge modification is carried out in either (but not both) a conventional Fab molecule(s) contained in an immunoactivating Fc domain binding molecule (e.g., as shown in Figures 2A-C, G-J) or a crossover Fab molecule(s) contained in an immunoactivating Fc domain binding molecule (e.g., as shown in Figures 2D-F, K-N). In certain embodiments, the charge modification is carried out in a conventional Fab molecule contained in an immunoactivating Fc domain binding molecule (specifically binding to a target cell antigen in certain embodiments).

[0218] CD3-bound immunoactivating Fc domain binding molecule In certain embodiments of the present invention, an immunoactivating Fc domain binding molecule can co-binding to at least one amino acid substitution of the first set described above and an Fc domain binding moiety that specifically binds to a target Fc domain, particularly CD3, which is an activated T cell antigen. The immunoactivating Fc domain binding molecule of the present invention is combined with a targeted antibody comprising an Fc domain which includes at least one amino acid substitution of the first set and at least one antigen-binding moiety capable of specific binding to an antigen on a target cell. In such embodiments, the immunoactivating Fc domain binding molecule can crosslink T cells and target cells by co-binding to the target Fc domain and the activated T cell antigen while the target antibody is bound to the target cell. In more detailed embodiments, such co-binding results in the lysis of target cells, particularly tumor cells. In one embodiment, such co-binding results in the activation of T cells. In other embodiments, such co-binding results in a cellular response of T lymphocytes, particularly cytotoxic T lymphocytes, selected from the group of proliferation, differentiation, cytokine secretion, release of cytotoxic effector molecules, cytotoxic activity, and expression of activation markers. In one embodiment, binding of an immunoactivating Fc domain-binding molecule to an activated T cell antigen, particularly CD3, without simultaneous crosslinking to target cells, does not result in T cell activation.

[0219] In one embodiment, an immunoactivating Fc domain-binding molecule combined with a targeted antibody can redirect the cytotoxic activity of T cells against target cells. In certain embodiments, this redirection is independent of MHC-mediated peptide antigen presentation by target cells and / or T cell specificity.

[0220] In particular, the T cells according to any embodiment of the present invention are cytotoxic T cells. In some embodiments, the T cells are CD4 + or CD8 + T cells, especially CD8 + These are T cells.

[0221] Therefore, in one aspect of the present invention, the immune activation portion is an antigen-binding portion capable of specific binding to activated T cell antigens, particularly CD3.

[0222] In one embodiment, the immunoactivating Fc domain binding molecule of the present invention comprises at least one Fab molecule (also referred herein as the "activated T cell antigen binding Fab molecule") that specifically binds to an activated T cell antigen. In a particular embodiment, the immunoactivating Fc domain binding molecule comprises one or fewer Fab molecules (or other Fab molecules) capable of specific binding to an activated T cell antigen. In one embodiment, the immunoactivating Fc domain binding molecule provides monovalent binding to an activated T cell antigen.

[0223] In certain embodiments, the Fab molecule that specifically binds to the activated T cell antigen is a crossover Fab molecule as described herein, i.e., a Fab molecule in which the variable domains VH and VL of the Fab heavy and light chains are exchanged / substituted with each other. In such embodiments, one or more Fab molecules that specifically bind to a target Fc domain containing at least one amino acid substitution of the first set are conventional Fab molecules. In embodiments where there are two or more Fab molecules that specifically bind to a target Fc domain contained in an immunoactivating Fc domain binding molecule, the Fab molecule that specifically binds to the activated T cell antigen is preferably a crossover Fab molecule, and the Fab molecule that specifically binds to the target Fc domain is a conventional Fab molecule.

[0224] In alternative embodiments, the Fab molecule that specifically binds to the activated T cell antigen is a conventional Fab molecule. In such embodiments, the Fab molecule that specifically binds to the target Fc domain is the crossover Fab molecule described herein, i.e., a Fab molecule in which the variable domains VH and VL of the Fab heavy and light chains are exchanged / substituted for each other.

[0225] In certain embodiments, the activated T cell antigen is CD3, particularly human CD3. In certain embodiments, the activated T cell antigen-binding Fab molecule is cross-reactive (i.e., specifically binds) to human and cynomolgus monkey CD3. In some embodiments, the activated T cell antigen is the epsilon subunit of CD3 (CD3 epsilon).

[0226] In some embodiments, the activated T cell antigen-binding Fab molecule specifically binds to CD3, particularly CD3 epsilon, and includes at least one heavy chain complementarity-determining region (CDR) selected from the group consisting of SEQ ID NOs. 35, 37, and 43, and at least one light chain CDR selected from the group consisting of SEQ ID NOs. 53, 54, and 55.

[0227] In one embodiment, the CD3-binding Fab molecule includes a heavy chain variable region containing heavy chain CDR1 of SEQ ID NO: 35, heavy chain CDR2 of SEQ ID NO: 37, and heavy chain CDR3 of SEQ ID NO: 43, and a light chain variable region containing light chain CDR1 of SEQ ID NO: 53, light chain CDR2 of SEQ ID NO: 54, and light chain CDR3 of SEQ ID NO: 55.

[0228] In one embodiment, the CD3-binding Fab molecule includes a heavy chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 49, and a light chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 56.

[0229] In one embodiment, the CD3-binding Fab molecule includes a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 49 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 56.

[0230] In some embodiments, the activated T cell antigen-binding Fab molecule specifically binds to CD3, particularly CD3 epsilon, and includes at least one heavy chain complementarity-determining region (CDR) selected from the group consisting of SEQ ID NOs: 34, 37, and 41, and at least one light chain CDR selected from the group consisting of SEQ ID NOs: 53, 54, and 55.

[0231] In one embodiment, the CD3-binding Fab molecule includes a heavy chain variable region containing heavy chain CDR1 of SEQ ID NO: 34, heavy chain CDR2 of SEQ ID NO: 37, and heavy chain CDR3 of SEQ ID NO: 41, and a light chain variable region containing light chain CDR1 of SEQ ID NO: 53, light chain CDR2 of SEQ ID NO: 54, and light chain CDR3 of SEQ ID NO: 55.

[0232] In one embodiment, the CD3-binding Fab molecule includes a heavy chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 47, and a light chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 56.

[0233] In one embodiment, the CD3-binding Fab molecule includes a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 47 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 56.

[0234] In some embodiments, the activated T cell antigen-binding Fab molecule specifically binds to CD3, particularly CD3 epsilon, and includes at least one heavy chain complementarity-determining region (CDR) selected from the group consisting of SEQ ID NO: 35, SEQ ID NO: 37, and SEQ ID NO: 176, and at least one light chain CDR selected from the group consisting of SEQ ID NO: 53, SEQ ID NO: 54, and SEQ ID NO: 55.

[0235] In one embodiment, the CD3-binding Fab molecule includes a heavy chain variable region containing heavy chain CDR1 of SEQ ID NO: 35, heavy chain CDR2 of SEQ ID NO: 37, and heavy chain CDR3 of SEQ ID NO: 176, and a light chain variable region containing light chain CDR1 of SEQ ID NO: 53, light chain CDR2 of SEQ ID NO: 54, and light chain CDR3 of SEQ ID NO: 55.

[0236] In one embodiment, the CD3-binding Fab molecule includes a heavy chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 177, and a light chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 56.

[0237] In one embodiment, the CD3-binding Fab molecule includes a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 177 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 56.

[0238] In one embodiment, the immunoactivating fragment crystallizable (Fc) domain binding molecule is, (a) A first light chain containing the amino acid sequence of Sequence ID No. 86, (b) A second light chain containing the amino acid sequence of Sequence ID No. 68, (c) The first heavy chain containing the amino acid sequence of SEQ ID NO: 87, (d) Second heavy chain containing the amino acid sequence of SEQ ID NO: 88 An immunoactivating fragment crystallizable (Fc) domain-binding molecule containing this domain is provided.

[0239] In one embodiment, the immunoactivating fragment crystallizable (Fc) domain binding molecule is, (a) A first light chain containing the amino acid sequence of Sequence ID No. 89, (b) A second light chain containing the amino acid sequence of Sequence ID No. 68, (c) The first heavy chain containing the amino acid sequence of SEQ ID NO: 90, (d) The second heavy chain containing the amino acid sequence of SEQ ID NO: 91 An immunoactivating fragment crystallizable (Fc) domain-binding molecule containing this domain is provided.

[0240] In one embodiment, the immunoactivating fragment crystallizable (Fc) domain binding molecule is, (a) A first light chain containing the amino acid sequence of Sequence ID No. 89, (b) A second light chain containing the amino acid sequence of SEQ ID NO: 70, (c) The first heavy chain containing the amino acid sequence of SEQ ID NO: 90, (d) The second heavy chain containing the amino acid sequence of SEQ ID NO: 91 An immunoactivating fragment crystallizable (Fc) domain-binding molecule containing this domain is provided.

[0241] In one embodiment, the immunoactivating fragment crystallizable (Fc) domain binding molecule is, (a) A first light chain containing the amino acid sequence of Sequence ID No. 89, (b) A second light chain containing the amino acid sequence of SEQ ID NO: 70, (c) The first heavy chain containing the amino acid sequence of SEQ ID NO: 90, (d) Second heavy chain containing the amino acid sequence of SEQ ID NO: 92 An immunoactivating fragment crystallizable (Fc) domain-binding molecule containing this domain is provided.

[0242] In one embodiment, the immunoactivating fragment crystallizable (Fc) domain binding molecule is, (a) A first light chain containing the amino acid sequence of SEQ ID NO: 93, (b) A second light chain containing the amino acid sequence of Sequence ID No. 68, (c) The first heavy chain containing the amino acid sequence of SEQ ID NO: 87, (d) Second heavy chain containing the amino acid sequence of SEQ ID NO: 88 An immunoactivating fragment crystallizable (Fc) domain-binding molecule containing this domain is provided.

[0243] In one embodiment, the immunoactivating fragment crystallizable (Fc) domain binding molecule is, (a) A first light chain having an amino acid sequence that is at least approximately 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 89, (b) A second light chain having an amino acid sequence that is at least approximately 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 70, (c) A first heavy chain comprising an amino acid sequence that is at least approximately 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 178, and (d) A second heavy chain containing an amino acid sequence that is at least approximately 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 179. An immunoactivating fragment crystallizable (Fc) domain-binding molecule is provided, which includes this component.

[0244] In one embodiment, the immunoactivating fragment crystallizable (Fc) domain binding molecule is, (a) A first light chain having an amino acid sequence that is at least approximately 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 89, (b) A second light chain having an amino acid sequence that is at least approximately 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 68, (c) A first heavy chain comprising an amino acid sequence that is at least approximately 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 178, and (d) A second heavy chain containing an amino acid sequence that is at least approximately 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 179. An immunoactivating fragment crystallizable (Fc) domain-binding molecule is provided, which includes this component.

[0245] In one embodiment, the immunoactivating fragment crystallizable (Fc) domain binding molecule is, (a) A first light chain having an amino acid sequence that is at least approximately 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 89, (b) A second light chain having an amino acid sequence that is at least approximately 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 180, (c) A first heavy chain comprising an amino acid sequence that is at least approximately 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 178, and (d) A second heavy chain containing an amino acid sequence that is at least approximately 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 179. An immunoactivating fragment crystallizable (Fc) domain-binding molecule is provided, which includes this component.

[0246] In one embodiment, the immunoactivating fragment crystallizable (Fc) domain binding molecule is, (a) FR-L1 containing the amino acid sequence of SEQ ID NO: 89; (b) A second light chain containing the amino acid sequence of SEQ ID NO: 70, (c) The first heavy chain containing the amino acid sequence of SEQ ID NO: 178, (d) Second heavy chain containing the amino acid sequence of SEQ ID NO: 179 and An immunoactivating fragment crystallizable (Fc) domain-binding molecule containing this domain is provided.

[0247] In one embodiment, the immunoactivating fragment crystallizable (Fc) domain binding molecule is, (a) A first light chain containing the amino acid sequence of Sequence ID No. 89, (b) A second light chain containing the amino acid sequence of Sequence ID No. 68, (c) The first heavy chain containing the amino acid sequence of SEQ ID NO: 178, (d) Second heavy chain containing the amino acid sequence of SEQ ID NO: 179 and An immunoactivating fragment crystallizable (Fc) domain-binding molecule containing this domain is provided.

[0248] In one embodiment, the immunoactivating fragment crystallizable (Fc) domain binding molecule is, (a) FR-L1 containing the amino acid sequence of SEQ ID NO: 89; (b) A second light chain containing the amino acid sequence of SEQ ID NO: 180, (c) The first heavy chain containing the amino acid sequence of SEQ ID NO: 187, (d) Second heavy chain containing the amino acid sequence of SEQ ID NO: 179 and An immunoactivating fragment crystallizable (Fc) domain-binding molecule containing this domain is provided.

[0249] In further embodiments, one of the above-described immunoactivating fragment crystallizable (Fc) domain binding molecules is provided, further comprising substitution at position P329 (numbered according to the Kabat EU index) with an amino acid selected from the list consisting of arginine (R), leucine (L), isoleucine (I), and alanine (A).

[0250] CD28-binding immunoactivating Fc domain binding molecule In certain embodiments of the present invention, an immunoactivating Fc domain binding molecule can co-bind with at least one amino acid substitution of the first set described above and an Fc domain binding moiety that specifically binds to a target Fc domain, particularly CD28, which is a costimulatory T cell antigen. The immunoactivating Fc domain binding molecule of the present invention is combined with a targeted antibody comprising an Fc domain comprising at least one amino acid substitution of the first set and at least one antigen-binding moiety capable of specific binding to an antigen on a target cell. In such embodiments, the immunoactivating Fc domain binding molecule can crosslink T cells and target cells by co-binding to the target Fc domain and the costimulatory T cell antigen while the target antibody is bound to the target cell. In more detailed embodiments, such co-binding results in the lysis of target cells, particularly tumor cells. In one embodiment, such co-binding results in the activation or increased activation of T cells. In other embodiments, such co-binding results in an increased cellular response of T lymphocytes, particularly cytotoxic T lymphocytes, selected from the group of proliferation, differentiation, cytokine secretion, release of cytotoxic effector molecules, cytotoxic activity, and expression of activation markers. In one embodiment, binding of an immunoactivating Fc domain-binding molecule to a co-stimulating T cell antigen, particularly CD28, without simultaneous crosslinking to target cells, does not result in (increased) T cell activation.

[0251] In one embodiment, an immunoactivating Fc domain-binding molecule combined with a targeted antibody can increase the cytotoxic activity of T cells against target cells. In a particular embodiment, the redirection is independent of MHC-mediated peptide antigen presentation by the target cells and / or T cell specificity.

[0252] In particular, the T cells according to any embodiment of the present invention are cytotoxic T cells. In some embodiments, the T cells are CD4 + or CD8 + T cells, especially CD8 + These are T cells.

[0253] Therefore, in one aspect of the present invention, the immune activation portion is an antigen-binding portion capable of specific binding to a costimulated T cell antigen, particularly CD28.

[0254] In one embodiment, the immune-activating Fc domain binding molecule of the present invention comprises at least one Fab molecule (also referred to herein as the "costimulatory T cell antigen binding Fab molecule") that specifically binds to a costimulatory T cell antigen. In a particular embodiment, the immune-activating Fc domain binding molecule comprises one or fewer Fab molecules (or other Fab molecules) capable of specific binding to a costimulatory T cell antigen. In one embodiment, the immune-activating Fc domain binding molecule provides monovalent binding to a costimulatory T cell antigen.

[0255] In certain embodiments, the Fab molecule that specifically binds to the costimulatory T cell antigen is a crossover Fab molecule as described herein, i.e., a Fab molecule in which the variable domains VH and VL of the Fab heavy and light chains are exchanged / substituted with each other. In such embodiments, one or more Fab molecules that specifically bind to a target Fc domain containing at least one amino acid substitution of the first set are conventional Fab molecules. In embodiments where there are two or more Fab molecules that specifically bind to a target Fc domain contained in an immunoactivating Fc domain binding molecule, the Fab molecule that specifically binds to the costimulatory T cell antigen is preferably a crossover Fab molecule, and the Fab molecule that specifically binds to the target Fc domain is a conventional Fab molecule.

[0256] In alternative embodiments, the Fab molecule that specifically binds to the costimulatory T cell antigen is a conventional Fab molecule. In such embodiments, the Fab molecule that specifically binds to the target Fc domain is the crossover Fab molecule described herein, i.e., a Fab molecule in which the variable domains VH and VL of the Fab heavy and light chains are exchanged / substituted with each other.

[0257] In certain embodiments, the costimulatory T cell antigen is CD28, particularly human CD28. In certain embodiments, the costimulatory T cell antigen-binding Fab molecule is cross-reactive (i.e., specifically binds) to human and cynomolgus monkey CD28.

[0258] In some embodiments, the co-stimulatory T cell antigen-binding Fab molecule specifically binds to CD28 and includes at least one heavy chain complementarity-determining region (CDR) selected from the group consisting of SEQ ID NOs. 94, 95, and 96, and at least one light chain CDR selected from the group consisting of SEQ ID NOs. 97, 98, and 99.

[0259] In one embodiment, the CD28-binding Fab molecule includes a heavy chain variable region containing heavy chain CDR1 of SEQ ID NO: 94, heavy chain CDR2 of SEQ ID NO: 95, and heavy chain CDR3 of SEQ ID NO: 96, and a light chain variable region containing light chain CDR1 of SEQ ID NO: 97, light chain CDR2 of SEQ ID NO: 98, and light chain CDR3 of SEQ ID NO: 99.

[0260] In some embodiments, the co-stimulatory T cell antigen-binding Fab molecule specifically binds to CD28 and includes at least one heavy chain complementarity-determining region (CDR) selected from the group consisting of SEQ ID NOs. 94, SEQ ID NOs. 95, and SEQ ID NOs. 102, and at least one light chain CDR selected from the group consisting of SEQ ID NOs. 103, SEQ ID NOs. 98, and SEQ ID NOs. 99.

[0261] In another embodiment, the CD28-binding Fab molecule includes a heavy chain variable region containing heavy chain CDR1 of SEQ ID NO: 94, heavy chain CDR2 of SEQ ID NO: 95, and heavy chain CDR3 of SEQ ID NO: 102, and a light chain variable region containing light chain CDR1 of SEQ ID NO: 103, light chain CDR2 of SEQ ID NO: 98, and light chain CDR3 of SEQ ID NO: 99.

[0262] In one embodiment, the CD28-binding Fab molecule includes a heavy chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 100, and a light chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 101.

[0263] In one embodiment, the CD28-binding Fab molecule includes a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 100 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 101.

[0264] In one embodiment, the CD28-binding Fab molecule includes the heavy chain variable region sequence of SEQ ID NO: 104 and the light chain variable region sequence of SEQ ID NO: 105.

[0265] In one embodiment, the CD28-binding Fab molecule includes a heavy chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 104, and a light chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 105.

[0266] In one embodiment, the CD28-binding Fab molecule includes a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 104 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 105.

[0267] In one embodiment, the CD28-binding Fab molecule includes the heavy chain variable region sequence of SEQ ID NO: 104 and the light chain variable region sequence of SEQ ID NO: 105.

[0268] In one embodiment, the immunoactivating fragment crystallizable (Fc) domain binding molecule is, (a) A first light chain containing the amino acid sequence of SEQ ID NO: 93, (b) A second light chain containing the amino acid sequence of SEQ ID NO: 106, (c) The first heavy chain containing the amino acid sequence of SEQ ID NO: 88, (d) Second heavy chain containing the amino acid sequence of SEQ ID NO: 107 An immunoactivating fragment crystallizable (Fc) domain-binding molecule containing this domain is provided.

[0269] In one embodiment, the immunoactivating fragment crystallizable (Fc) domain binding molecule is, (a) A first light chain containing the amino acid sequence of SEQ ID NO: 93, (b) A second light chain containing the amino acid sequence of Sequence ID No. 108, (c) The first heavy chain containing the amino acid sequence of SEQ ID NO: 88, (d) The second heavy chain containing the amino acid sequence of SEQ ID NO: 109 An immunoactivating fragment crystallizable (Fc) domain-binding molecule containing this domain is provided.

[0270] In one embodiment, the immunoactivating fragment crystallizable (Fc) domain binding molecule is, (a) A first light chain containing the amino acid sequence of Sequence ID No. 89, (b) A second light chain containing the amino acid sequence of Sequence ID No. 108, (c) The first heavy chain containing the amino acid sequence of SEQ ID NO: 90, (d) The second heavy chain containing the amino acid sequence of SEQ ID NO: 109 An immunoactivating fragment crystallizable (Fc) domain-binding molecule containing this domain is provided.

[0271] In one embodiment, the immunoactivating fragment crystallizable (Fc) domain binding molecule is, (a) A first light chain containing the amino acid sequence of Sequence ID No. 110, (b) A second light chain containing the amino acid sequence of SEQ ID NO: 111, (c) The first heavy chain containing the amino acid sequence of SEQ ID NO: 112, (d) Second heavy chain containing the amino acid sequence of SEQ ID NO: 113 An immunoactivating fragment crystallizable (Fc) domain-binding molecule containing this domain is provided.

[0272] In one embodiment, the immunoactivating fragment crystallizable (Fc) domain binding molecule is, (a) A first light chain containing the amino acid sequence of Sequence ID No. 89, (b) A second light chain containing the amino acid sequence of Sequence ID No. 108, (c) The first heavy chain containing the amino acid sequence of SEQ ID NO: 90, (d) Second heavy chain containing the amino acid sequence of SEQ ID NO: 114 An immunoactivating fragment crystallizable (Fc) domain-binding molecule containing this domain is provided.

[0273] In one embodiment, the immunoactivating fragment crystallizable (Fc) domain binding molecule is, (a) A first light chain containing the amino acid sequence of Sequence ID No. 89, (b) A second light chain containing the amino acid sequence of Sequence ID No. 108, (c) The first heavy chain containing the amino acid sequence of SEQ ID NO: 90, (d) Second heavy chain containing the amino acid sequence of SEQ ID NO: 115 An immunoactivating fragment crystallizable (Fc) domain-binding molecule containing this domain is provided.

[0274] In further embodiments, one of the above-described immunoactivating fragment crystallizable (Fc) domain binding molecules is provided, further comprising substitution at position P329 (numbered according to the Kabat EU index) with an amino acid selected from the list consisting of arginine (R), leucine (L), isoleucine (I), and alanine (A).

[0275] 4-1BB-conjugated immune-activating Fc domain binding molecule In certain embodiments of the present invention, an immunoactivating Fc domain binding molecule can co-binding to a target Fc domain containing at least one amino acid substitution of the first set described above and to an Fc domain binding moiety that specifically binds to 4-1BB. Thus, in one aspect of the present invention, the immunoactivating moiety is an antigen-binding moiety capable of specific binding to a costimulatory T cell antigen, particularly 4-1BB. The immunoactivating Fc domain binding molecule of the present invention is combined with a targeted antibody comprising an Fc domain containing at least one amino acid substitution of the first set and at least one antigen-binding moiety capable of specific binding to an antigen on a target cell. In such embodiments, the immunoactivating Fc domain binding molecule can crosslink T cells and target cells by co-binding to the target Fc domain and the costimulatory T cell antigen while the target antibody is bound to the target cell. In more detailed embodiments, such co-binding results in the lysis of target cells, particularly tumor cells. In one embodiment, such co-binding results in the activation or increased activation of T cells. In other embodiments, such co-binding results in an increased cellular response of T lymphocytes, particularly cytotoxic T lymphocytes, selected from the group of proliferation, differentiation, cytokine secretion, release of cytotoxic effector molecules, and expression of cytotoxic activity and activation markers. In one embodiment, binding of an immunoactivating Fc domain-binding molecule to a co-stimulating T cell antigen, particularly 4-1BB, without co-crosslinking to target cells does not result in increased T cell activation.

[0276] In one embodiment, an immunoactivating Fc domain-binding molecule combined with a targeted antibody can increase the cytotoxic activity of T cells against target cells. In a particular embodiment, the redirection is independent of MHC-mediated peptide antigen presentation by the target cells and / or T cell specificity.

[0277] In particular, the T cells according to any embodiment of the present invention are cytotoxic T cells. In some embodiments, the T cells are CD4 + or CD8 + T cells, especially CD8 + These are T cells.

[0278] In one embodiment, the immune-activating Fc domain binding molecule of the present invention comprises at least one Fab molecule (also referred to herein as the "costimulatory T cell antigen binding Fab molecule") that specifically binds to a costimulatory T cell antigen. In a particular embodiment, the immune-activating Fc domain binding molecule comprises one or fewer Fab molecules (or other Fab molecules) capable of specific binding to a costimulatory T cell antigen. In one embodiment, the immune-activating Fc domain binding molecule provides monovalent binding to the costimulatory antigen.

[0279] In certain embodiments, the Fab molecule that specifically binds to the costimulatory T cell antigen is a crossover Fab molecule as described herein, i.e., a Fab molecule in which the variable domains VH and VL of the Fab heavy and light chains are exchanged / substituted with each other. In such embodiments, one or more Fab molecules that specifically bind to a target Fc domain containing at least one amino acid substitution of the first set are conventional Fab molecules. In embodiments where there are two or more Fab molecules that specifically bind to a target Fc domain contained in an immunoactivating Fc domain binding molecule, the Fab molecule that specifically binds to the costimulatory T cell antigen is preferably a crossover Fab molecule, and the Fab molecule that specifically binds to the target Fc domain is a conventional Fab molecule.

[0280] In alternative embodiments, the Fab molecule that specifically binds to the costimulatory T cell antigen is a conventional Fab molecule. In such embodiments, the Fab molecule that specifically binds to the target Fc domain is the crossover Fab molecule described herein, i.e., a Fab molecule in which the variable domains VH and VL of the Fab heavy and light chains are exchanged / substituted with each other.

[0281] In certain embodiments, the costimulatory T cell antigen is 4-1BB, specifically human CD28. In certain embodiments, the costimulatory T cell antigen-binding Fab molecule is cross-reactive (i.e., specifically binds) to human and cynomolgus monkey 4-1BB.

[0282] In some embodiments, the co-stimulatory T cell antigen-binding Fab molecule specifically binds to 4-1BB and includes at least one heavy chain complementarity-determining region (CDR) selected from the group consisting of SEQ ID NOs: 133, 134, and 135, and at least one light chain CDR selected from the group consisting of SEQ ID NOs: 136, 137, and 138.

[0283] In one embodiment, the 4-1BB-bound Fab molecule includes a heavy chain variable region containing heavy chain CDR1 of SEQ ID NO: 133, heavy chain CDR2 of SEQ ID NO: 134, and heavy chain CDR3 of SEQ ID NO: 135, and a light chain variable region containing light chain CDR1 of SEQ ID NO: 136, light chain CDR2 of SEQ ID NO: 137, and light chain CDR3 of SEQ ID NO: 138.

[0284] In one embodiment, the 4-1BB-binding Fab molecule includes a heavy chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 139, and a light chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 140.

[0285] In one embodiment, the 4-1BB-binding Fab molecule includes a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 139 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 140.

[0286] In one embodiment, the CD28-binding Fab molecule includes the heavy chain variable region sequence of SEQ ID NO: 139 and the light chain variable region sequence of SEQ ID NO: 140.

[0287] In one embodiment, the immunoactivating fragment crystallizable (Fc) domain binding molecule is, (a) A first light chain containing the amino acid sequence of SEQ ID NO: 141, (b) A second light chain containing the amino acid sequence of SEQ ID NO: 142, (c) The first heavy chain containing the amino acid sequence of SEQ ID NO: 143, (d) Second heavy chain containing the amino acid sequence of SEQ ID NO: 144 and An immunoactivating fragment crystallizable (Fc) domain-binding molecule containing this domain is provided.

[0288] In one embodiment, the immunoactivating fragment crystallizable (Fc) domain binding molecule is, (a) A first light chain containing the amino acid sequence of Sequence ID No. 110, (b) A second light chain containing the amino acid sequence of SEQ ID NO: 142, (c) The first heavy chain containing the amino acid sequence of SEQ ID NO: 145, (d) Second heavy chain containing the amino acid sequence of SEQ ID NO: 144 and An immunoactivating fragment crystallizable (Fc) domain-binding molecule containing this domain is provided.

[0289] In further embodiments, one of the above-described immunoactivating fragment crystallizable (Fc) domain binding molecules is provided, further comprising substitution at position P329 (numbered according to the Kabat EU index) with an amino acid selected from the list consisting of arginine (R), leucine (L), isoleucine (I), and alanine (A).

[0290] Immune-activating Fc domain binding molecules containing cytokines In one embodiment, the immune-activating portion is a cytokine. In one embodiment, the cytokine is selected from the group consisting of IL2, IL7, IL15, IL18, IFNa, and IFNg. In such specific embodiments, the immune-activating fragment crystallizable (Fc) domain-binding molecule of the present invention includes a variant IL-2 polypeptide having properties advantageous for immunotherapy. In particular, pharmacological properties of IL-2 that contribute to toxicity but are not essential for the efficacy of IL-2 are removed in the variant IL-2 polypeptide. Such variant IL-2 polypeptides are described in detail in International Publication No. 2012 / 107417, which is incorporated in whole hereby for reference. As described above, different forms of IL-2 receptors consist of different subunits and exhibit different affinities to IL-2. The moderate-affinity IL-2 receptor consists of β and γ receptor subunits, is expressed in resting effector cells, and is sufficient for IL-2 signaling. The high-affinity IL-2 receptor further includes the α-subunit of the receptor and regulatory T(T) reg ) Primarily expressed in cells and activated effector cells, and upon engagement by IL-2, T reg Cell-mediated immunosuppression or activation-induced cell death (AICD) can be promoted. Therefore, although we do not wish to be constrained by theory, a decrease or absence of IL-2 affinity for the α-subunit of the IL-2 receptor should reduce IL-2-induced downregulation of effector cell function by regulatory T cells and the progression of tumor resistance through the AICD process. On the other hand, maintaining affinity for moderate-affinity IL-2 receptors should preserve IL-2-induced proliferation and activation of effector cells such as NK cells and T cells.

[0291] The mutant interleukin-2 (IL-2) polypeptide contained in the immunoactivating fragment crystallizable (Fc) domain binding molecule according to the present invention contains at least one amino acid mutation that eliminates or reduces the affinity of the mutant IL-2 polypeptide to the α-subunit of the IL-2 receptor, while preserving the affinity of the mutant IL-2 polypeptide to the moderate-affinity IL-2 receptor, compared to each wild-type IL-2 polypeptide.

[0292] Mutants of human IL-2 (hIL-2) with reduced affinity for CD25 may be created, for example, by amino acid substitutions at amino acid positions 35, 38, 42, 43, 45, or 72, or combinations thereof (numbering relative to the human IL-2 sequence, SEQ ID NO: 166). Examples of amino acid substitutions include K35E, K35A, R38A, R38E, R38N, R38F, R38S, R38L, R38G, R38Y, R38W, F42L, F42A, F42G, F42S, F42T, F42Q, F42E, F42N, F42D, F42R, F42K, K43E, Y45A, Y45G, Y45S, Y45T, Y45Q, Y45E, Y45N, Y45D, Y45R, Y45K, L72G, L72A, L72S, L72T, L72Q, L72E, L72N, L72D, L72R, and L72K. Specific IL-2 variants useful for the immunoactivating fragment crystallizable (Fc) domain binding molecule of the present invention include amino acid mutations at amino acid positions corresponding to residues 42, 45, or 72 of human IL-2, or combinations thereof. In one embodiment, such amino acid mutations are amino acid substitutions selected from the group F42A, F42G, F42S, F42T, F42Q, F42E, F42N, F42D, F42R, F42K, Y45A, Y45G, Y45S, Y45T, Y45Q, Y45E, Y45N, Y45D, Y45R, Y45K, L72G, L72A, L72S, L72T, L72Q, L72E, L72N, L72D, L72R, and L72K, more specifically, amino acid substitutions selected from the group F42A and Y45A and L72G. These mutants exhibit substantially similar binding affinity to the moderate-affinity IL-2 receptor, and show significantly reduced affinity for the α-subunit of the IL-2 receptor and the high-affinity IL-2 receptor compared to the wild-type IL-2 mutant.

[0293] Other characteristics of useful mutants may include the ability to induce proliferation of IL-2 receptor-containing T cells and / or NK cells, the ability to induce IL-2 signaling in IL-2 receptor-containing T cells and / or NK cells, the ability to generate interferon (IFN)-γ as a secondary cytokine by NK cells, reduced ability to induce secondary cytokine production (particularly IL-10 and TNF-α) by peripheral blood mononuclear cells (PBMCs), reduced ability to activate regulatory T cells, reduced ability to induce apoptosis in T cells, and a reduced in vivo toxicity profile.

[0294] Specific mutant IL-2 polypeptides useful in the present invention contain three amino acid mutations that preserve the affinity of the mutant IL-2 polypeptide to the α-subunit of the IL-2 receptor, but to a moderate affinity IL-2 receptor, while having little to no affinity for the α-subunit of the IL-2 receptor. In one embodiment, these three amino acid mutations are located at positions corresponding to residues 42, 45, and 72 of human IL-2. In one embodiment, these three amino acid mutations are amino acid substitutions. In one embodiment, the three amino acid mutation is an amino acid substitution selected from the group F42A, F42G, F42S, F42T, F42Q, F42E, F42N, F42D, F42R, F42K, Y45A, Y45G, Y45S, Y45T, Y45Q, Y45E, Y45N, Y45D, Y45R, Y45K, L72G, L72A, L72S, L72T, L72Q, L72E, L72N, L72D, L72R, and L72K. In a specific embodiment, the three amino acid mutation is the amino acid substitution F42A, Y45A, and L72G (numbering for the human IL-2 sequence of SEQ ID NO: 166).

[0295] In certain embodiments, the amino acid mutation reduces the affinity of the mutant IL-2 polypeptide to the α-subunit of the IL-2 receptor by at least one-fifth, more specifically at least one-tenth, and more specifically at least one-twenty-fifth. In embodiments where there are more than one amino acid mutations that reduce the affinity of the mutant IL-2 polypeptide to the α-subunit of the IL-2 receptor, the combination of these amino acid mutations may reduce the affinity of the mutant IL-2 polypeptide to the α-subunit of the IL-2 receptor by at least one-thirtieth, at least one-fiftieth, or even further at least one-hundredth. In one embodiment, the amino acid mutation, or combination of amino acid mutations, eliminates the affinity of the mutant IL-2 polypeptide to the α-subunit of the IL-2 receptor so that binding is not detectable by surface plasmon resonance.

[0296] Substantially similar binding to moderate affinity receptors (i.e., conservation of the affinity of the mutant IL-2 polypeptide to the receptor) is achieved when the IL-2 mutant exhibits an affinity greater than approximately 70% of the wild-type affinity of the IL-2 mutant to moderate affinity IL-2 receptors. The IL-2 mutants of the present invention may exhibit affinities greater than approximately 80% and even greater than approximately 90% of such affinities.

[0297] Combining the removal of O-glycosylation of IL-2 with a reduction in the affinity of IL-2 for the α-subunit of the IL-2 receptor can yield an IL-2 protein with improved properties. For example, the absence of the O-glycosylation site results in a more homogeneous product when the mutant IL-2 polypeptide is expressed in mammalian cells such as CHO cells or HEK cells.

[0298] Therefore, in certain embodiments, the mutant IL-2 polypeptide includes further amino acid mutations that result in the absence of an O-glycosylation site of IL-2 at the position corresponding to residue 3 of human IL-2. In one embodiment, the further amino acid mutation that eliminates the O-glycosylation site of IL-2 at the position corresponding to residue 3 of human IL-2 is an amino acid substitution. Exemplary amino acid substitutions include T3A, T3G, T3Q, T3E, T3N, T3D, T3R, T3K, and T3P. In a specific embodiment, the further amino acid mutation is the amino acid substitution T3A.

[0299] In certain embodiments, the mutant IL-2 polypeptide is essentially a full-length IL-2 molecule. In certain embodiments, the mutant IL-2 polypeptide is a human IL-2 molecule. In one embodiment, the mutant IL-2 polypeptide includes the sequence of SEQ ID NO: 166 having at least one amino acid mutation that eliminates or reduces the affinity of the mutant IL-2 polypeptide to the α-subunit of the IL-2 receptor compared to the IL-2 polypeptide containing SEQ ID NO: 166 without the above mutation, but preserves the affinity of the mutant IL-2 polypeptide to a moderate-affinity IL-2 receptor. In another embodiment, the mutant IL-2 polypeptide includes the sequence of SEQ ID NO: 167 having at least one amino acid mutation that eliminates or reduces the affinity of the mutant IL-2 polypeptide to the α-subunit of the IL-2 receptor compared to the IL-2 polypeptide containing SEQ ID NO: 167 without the above mutation, but preserves the affinity of the mutant IL-2 polypeptide to a moderate-affinity IL-2 receptor.

[0300] In specific embodiments, the mutant IL-2 polypeptide can induce one or more cellular responses selected from the group consisting of: proliferation of activated T lymphocytes, differentiation of activated T lymphocytes, cytotoxic T cell (CTL) activity, increase in activated B cells, differentiation of activated B cells, proliferation of natural killer (NK) cells, differentiation of NK cells, cytokine secretion by activated T cells or NK cells, and NK / lymphocyte-activated killer (LAK) antitumor cytotoxicity.

[0301] In one embodiment, the mutant IL-2 polypeptide has a reduced ability to induce IL-2 signaling in regulatory T cells compared to the wild-type IL-2 polypeptide. In one embodiment, the mutant IL-2 polypeptide induces less activation-induced cell death (AICD) in T cells compared to the wild-type IL-2 polypeptide. In one embodiment, the mutant IL-2 polypeptide has a reduced in vivo toxicity profile compared to the wild-type IL-2 polypeptide. In one embodiment, the mutant IL-2 polypeptide has a longer serum half-life compared to the wild-type IL-2 polypeptide.

[0302] The specific variant IL-2 polypeptide useful in this invention contains four amino acid substitutions at positions corresponding to residues 3, 42, 45, and 72 of human IL-2. The specific amino acid substitutions are T3A, F42A, Y45A, and L72G. As shown in International Publication No. 2012 / 107417, the quadruple variant IL-2 polypeptide does not show detectable binding to CD25 and exhibits reduced ability to induce apoptosis in T cells. reg It exhibits reduced ability to induce IL-2 signaling in cells and a reduced in vivo toxicity profile. However, it retains the ability to activate IL-2 signaling in effector cells, induce effector cell proliferation, and generate IFN-γ as a secondary cytokine by NK cells.

[0303] Furthermore, as described in International Publication No. 2012 / 107417, the mutant IL-2 polypeptide possesses even more advantageous characteristics, such as reduced surface hydrophobicity, good stability, and good expression yield. Unexpectedly, the mutant IL-2 polypeptide also has a longer serum half-life compared to wild-type IL-2.

[0304] In addition to having mutations in the region of IL-2 that forms the interface of CD25 or the glycosylation site, IL-2 mutants useful in the present invention may also have one or more mutations in the amino acid sequence outside these regions. Such further mutations in human IL-2 may provide further advantages such as increased expression or stability. For example, the cysteine ​​at position 125 may be replaced with a neutral amino acid such as serine, alanine, threonine, or valine, resulting in C125S IL-2, C125A IL-2, C125T IL-2, or C125V IL-2, respectively, as described in U.S. Patent No. 4,518,584. As described herein, deletion of the N-terminal alanine residue of IL-2 may yield mutants such as des-A1 C125S or des-A1 C125A. Alternatively, or in combination therewith, the IL-2 variants may contain mutations such that the methionine normally present at position 104 of wild-type human IL-2 is replaced with a neutral amino acid such as alanine (see U.S. Patent No. 5,206,344). The resulting variants, for example, des-A1 M104A IL-2, des-A1 M104A C125S IL-2, M104A IL-2, M104A C125A IL-2, des-A1 M104A C125A IL-2, or M104A C125S IL-2 (these and other variants are found in U.S. Patent No. 5,116,943 and Weiger et al., Eur J Biochem 180, 295-300 (1989)), may be used in combination with the specific IL-2 variants of the present invention.

[0305] Therefore, in certain embodiments, the mutant IL-2 polypeptide contains a further amino acid mutation at the position corresponding to residue 125 of human IL-2. In one embodiment, the further amino acid mutation is the amino acid substitution C125A.

[0306] Those skilled in the art can determine which further mutations may provide further advantages to the objectives of the present invention. For example, amino acid mutations in the IL-2 sequence that reduce or eliminate the affinity of IL-2 to the moderate affinity IL-2 receptor, such as D20T, N88R, or Q126D (see, for example, U.S. Patent Application Publication No. 2007 / 0036752), may not be appropriate to include in the variant IL-2 polypeptide according to the present invention.

[0307] In one embodiment, the mutant IL-2 polypeptide contains 12 or fewer amino acid mutations, 11 or fewer, 10 or fewer, 9 or fewer, 8 or fewer, 7 or fewer, 6 or fewer, or 5 or fewer, compared to the corresponding wild-type IL-2 sequence, e.g., the human IL-2 sequence of SEQ ID NO: 166. In a particular embodiment, the mutant IL-2 polypeptide contains 5 or fewer amino acid mutations compared to the corresponding wild-type IL-2 sequence, e.g., the human IL-2 sequence of SEQ ID NO: 166.

[0308] In one embodiment, the mutant IL-2 polypeptide contains the sequence of SEQ ID NO: 167.

[0309] In one embodiment, the present invention relates to an immunoactivating fragment crystallizable (Fc) domain binding molecule containing mutant IL-2, wherein mutant IL-2 is (a) an Fc domain binding moiety that specifically binds to a target Fc domain comprising at least one amino acid substitution of the first set described herein, (b) An immunoactivating moiety which is a mutant IL-2 polypeptide, wherein the mutant IL-2 polypeptide is a human IL-2 molecule containing amino acid substitutions F42A, Y45A and L72G (numbering for the human IL-2 sequence of SEQ ID NO: 166), (c) A half-life extension Fc domain comprising a first subunit and a second subunit capable of stable association as described herein, Includes, The Fc domain binding portion does not specifically bind to the half-life extended Fc domain, and provides an immunoactivating fragment crystallizable (Fc) domain binding molecule containing mutant IL-2.

[0310] In one embodiment, the present invention relates to an immunoactivating fragment crystallizable (Fc) domain binding molecule containing mutant IL-2, wherein mutant IL-2 is (a) an Fc domain binding moiety that specifically binds to a target Fc domain comprising at least one amino acid substitution of the first set described herein, (b) An immunoactivating moiety which is a mutant IL-2 polypeptide, wherein the mutant IL-2 polypeptide is a human IL-2 molecule containing amino acid substitutions T3A, F42A, Y45A, L72G and C125A (numbering for the human IL-2 sequence of SEQ ID NO: 166), (c) A half-life extension Fc domain comprising a first subunit and a second subunit capable of stable association as described herein, Includes, The Fc domain binding portion does not specifically bind to the half-life extended Fc domain, and provides an immunoactivating fragment crystallizable (Fc) domain binding molecule containing mutant IL-2.

[0311] In one embodiment, the present invention relates to an immunoactivating fragment crystallizable (Fc) domain binding molecule containing mutant IL-2, wherein mutant IL-2 is (a) an Fc domain binding moiety that specifically binds to a target Fc domain comprising at least one amino acid substitution of the first set described herein, (b) An immunoactivating moiety which is a mutant IL-2 polypeptide, wherein the mutant IL-2 polypeptide contains the amino acid sequence of SEQ ID NO: 167, (c) A half-life extension Fc domain comprising a first subunit and a second subunit capable of stable association as described herein, Includes, The Fc domain binding portion does not specifically bind to the half-life extended Fc domain, and provides an immunoactivating fragment crystallizable (Fc) domain binding molecule containing mutant IL-2.

[0312] In any of the embodiments described above, the mutant IL-2 polypeptide can be fused via a linker peptide to the carboxy-terminal amino acids of one or both subunits of the half-life extension Fc domain at its amino-terminal amino acid.

[0313] In one embodiment, an immunoactivating fragment crystallizable (Fc) domain binding molecule, (a) A light chain containing the amino acid sequence of Sequence ID No. 86, (c) The first heavy chain containing the amino acid sequence of SEQ ID NO: 116, (d) Second heavy chain containing the amino acid sequence of SEQ ID NO: 88 An immunoactivating fragment crystallizable (Fc) domain-binding molecule containing this domain is provided.

[0314] In one embodiment, an immunoactivating fragment crystallizable (Fc) domain binding molecule, (a) A light chain containing the amino acid sequence of Sequence ID No. 15, (c) The first heavy chain containing the amino acid sequence of SEQ ID NO: 116, (d) The second heavy chain containing the amino acid sequence of SEQ ID NO: 90 An immunoactivating fragment crystallizable (Fc) domain-binding molecule containing this domain is provided.

[0315] In a preferred embodiment, the immunoactivating fragment crystallizable (Fc) domain-binding molecule is combined with a targeted antibody capable of specific binding to T cell antigens, particularly CD8 or PD-1. In a preferred embodiment, the targeted antibody is capable of specific binding to PD-1. Such a combination is useful for cis-activation of T cells (see Figure 44). In a specific embodiment, the immunoactivating fragment crystallizable (Fc) domain-binding molecule is combined with a targeted antibody comprising a first light chain containing the amino acid sequence of SEQ ID NO: 160 and a heavy chain containing the amino acid sequence of SEQ ID NO: 161.

[0316] In further embodiments, one of the above-described immunoactivating fragment crystallizable (Fc) domain binding molecules is provided, further comprising substitution at position P329 (numbered according to the Kabat EU index) with an amino acid selected from the list consisting of arginine (R), leucine (L), isoleucine (I), and alanine (A).

[0317] 4-1BBL trimer-containing immune activation Fc domain binding molecule In one aspect of the present invention, the immunoactivating moiety is a costimulatory T cell ligand, particularly 4-1BBL. Therefore, in another aspect, the present invention also provides a novel 4-1BBL trimer-containing immunoactivating Fc domain-binding molecule.

[0318] In a first aspect, the present invention relates to an immunoactivating fragment crystallizable (Fc) domain binding molecule, (a) an Fc domain binding moiety that specifically binds to the target Fc domain described herein, (b) A first and second polypeptide linked to each other by a disulfide bond (the immunoactivating Fc domain binding molecule is characterized in that the first polypeptide comprises two external domains or fragments thereof of 4-1BBL linked to each other by a peptide linker, and the second polypeptide comprises one external domain or fragment thereof of 4-1BBL), (c) A half-life extension Fc domain comprising a first subunit and a second subunit capable of stable association as described herein, Includes, The Fc domain binding portion provides an immunoactivating fragment crystallizable (Fc) domain binding molecule that does not specifically bind to half-life extension Fc domains.

[0319] In a further embodiment, an immunoactivating fragment crystallizable (Fc) domain binding molecule as previously defined herein, (a) an Fc domain binding moiety that specifically binds to the target Fc domain described herein, (b) The first and second polypeptides linked to each other by a disulfide bond (the immune-activating Fc domain binding molecule, (i) The first polypeptide comprises a CH1 or CL domain, and the second polypeptide comprises a CL or CH1 domain, wherein the second polypeptide is linked to the first polypeptide by a disulfide bond between the CH1 and CL domains, and the first polypeptide comprises two external domains or fragments thereof of 4-1BBL connected to each other and to the CH1 or CL domain by a peptide linker, and the second polypeptide comprises one external domain or fragment thereof of 4-1BBL connected to the CL or CH1 domain of the polypeptide via a peptide linker, or (ii) The first polypeptide comprises a CH3 domain, and the second polypeptide comprises a CH3 domain, wherein the first polypeptide comprises two external domains or fragments thereof of 4-1BBL connected to each other and to the C-terminus of the CH3 domain by a peptide linker, and the second polypeptide comprises only one external domain or fragment thereof of 4-1BBL connected to the C-terminus of the CH3 domain of the polypeptide via a peptide linker, or (iii) a first polypeptide comprising a VH-CL or VL-CH1 domain, a second polypeptide comprising a VL-CH1 domain or a VH-CL domain, wherein the second polypeptide is linked to the first polypeptide by a disulfide bond between the CH1 and CL domains, the first polypeptide comprising two 4-1BBL external domains or fragments thereof linked to each other and to VH or VL by a peptide linker, and the second polypeptide comprising one external domain or fragment thereof of the TNF ligan family member linked to the VL or VH of the polypeptide via a peptide linker. (c) A half-life extension Fc domain comprising a first subunit and a second subunit capable of stable association as described herein, Includes, An immunoactivating fragment crystallizable (Fc) domain binding molecule is provided, in which the Fc domain binding portion does not specifically bind to the half-life extension Fc domain.

[0320] In another embodiment, an immunoactivating fragment crystallizable (Fc) domain binding molecule as previously defined herein, (a) an Fc domain binding moiety that specifically binds to the target Fc domain described herein, (b) The first and second polypeptides linked to each other by a disulfide bond (the immune-activating Fc domain binding molecule, (i) The first polypeptide comprises a CH1 or CL domain, and the second polypeptide comprises a CL or CH1 domain, wherein the second polypeptide is linked to the first polypeptide by a disulfide bond between the CH1 and CL domains, and the first polypeptide comprises two external domains or fragments thereof of 4-1BBL connected to each other and to the CH1 or CL domain by a peptide linker, and the second polypeptide comprises one external domain or fragment thereof of 4-1BBL connected to the CL or CH1 domain of the polypeptide via a peptide linker, or (ii) The first polypeptide comprises a CH3 domain, and the second polypeptide comprises a CH3 domain, wherein the first polypeptide comprises two external domains or fragments thereof of 4-1BBL connected to each other and to the C-terminus of the CH3 domain by a peptide linker, and the second polypeptide comprises only one external domain or fragment thereof of 4-1BBL connected to the C-terminus of the CH3 domain of the polypeptide via a peptide linker, (c) A half-life extension Fc domain comprising a first subunit and a second subunit capable of stable association as described herein, Includes, An immunoactivating fragment crystallizable (Fc) domain binding molecule is provided, in which the Fc domain binding portion does not specifically bind to the half-life extension Fc domain.

[0321] In one embodiment, the external domain of 4-1BBL includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 117, 118, 119, 120, 121, 122, 123, and 124, particularly the amino acid sequence of SEQ ID NO: 117 or 121. More specifically, the external domain of 4-1BBL includes the amino acid sequence of SEQ ID NO: 117 or 121. Most specifically, the external domain of 4-1BBL includes the amino acid sequence of SEQ ID NO: 121. In particular, an immunoactivating fragment crystallizable (Fc) domain-binding molecule, as previously defined herein, is provided, in which all three external domains or fragments of 4-1BBL are identical.

[0322] In a further embodiment, the immunoactivating fragment crystallizable (Fc) domain binding molecule of the present invention is (a) an Fc domain binding moiety that specifically binds to the target Fc domain described herein, (b) A first and second polypeptide linked to each other by a disulfide bond (the antigen-binding molecule is characterized in that the first polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs. 125, SEQ ID NOs. 126, SEQ ID NOs. 127 and SEQ ID NOs. 128, and the second polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs. 117, SEQ ID NOs. 121, SEQ ID NOs. 119 and SEQ ID NOs. 120), (c) A half-life extension Fc domain comprising a first subunit and a second subunit capable of stable association as described herein, Includes, The Fc domain binding portion does not specifically bind to the half-life extension Fc domain.

[0323] In one embodiment, the immunoactivating fragment crystallizable (Fc) domain binding molecule of the present invention is (a) an Fc domain binding moiety that specifically binds to the target Fc domain described herein, (b) The first and second polypeptides linked to each other by a disulfide bond (the antigen-binding molecule consists of the first polypeptide containing the amino acid sequence of SEQ ID NO: 126 and the second polypeptide containing the amino acid sequence of SEQ ID NO: 121), (c) A half-life extension Fc domain comprising a first subunit and a second subunit capable of stable association as described herein, Includes, The Fc domain binding portion does not specifically bind to the half-life extension Fc domain.

[0324] In a further embodiment, the immunoactivating fragment crystallizable (Fc) domain binding molecule of the present invention is (a) an Fc domain binding moiety that specifically binds to the target Fc domain described herein, (b) The first and second polypeptides linked to each other by a disulfide bond (the antigen-binding molecule consists of the first polypeptide containing the amino acid sequence of SEQ ID NO: 125 and the second polypeptide containing the amino acid sequence of SEQ ID NO: 117), (c) A half-life extension Fc domain comprising a first subunit and a second subunit capable of stable association as described herein, Includes, The Fc domain binding portion does not specifically bind to the half-life extension Fc domain.

[0325] In another embodiment, the immunoactivating fragment crystallizable (Fc) domain binding molecule of the present invention is (a) an Fc domain binding moiety that specifically binds to the target Fc domain described herein, (b) A first polypeptide comprising a CH1 domain and a CL domain, and a second polypeptide comprising a CL domain and a CH1 domain, wherein the second polypeptide is linked to the first polypeptide by a disulfide bond between the CH1 domain and the CL domain. The antigen-binding molecule is characterized in that the first polypeptide comprises two external domains or fragments thereof of 4-1BBL connected to each other and to the CH1 or CL domain by a peptide linker, and the second polypeptide comprises only one external domain or fragment thereof of 4-1BBL connected to the CL or CH1 domain of the polypeptide by a peptide linker.

[0326] In one embodiment, an immunoactivating fragment crystallizable (Fc) domain binding molecule, (a) an Fc domain binding moiety that specifically binds to the target Fc domain described herein, (b) comprising a first polypeptide containing a CH1 domain and a second polypeptide containing a CL domain, wherein the second polypeptide is linked to the first polypeptide by a disulfide bond between the CH1 domain and the CL domain. The antigen-binding molecule is provided as an immunoactivating fragment crystallizable (Fc) domain-binding molecule, characterized in that the first polypeptide comprises two external domains or fragments thereof of 4-1BBL linked to each other and to the CH1 domain by a peptide linker, and the second polypeptide comprises one ectodomain or fragment thereof of 4-1BBL linked to the CL domain of the polypeptide via a peptide linker.

[0327] In another aspect, the present invention relates to an immunoactivating fragment crystallizable (Fc) domain binding molecule, (a) an Fc domain binding moiety that specifically binds to the target Fc domain described herein, (b) A first and second polypeptide linked to each other by a disulfide bond (the antigen-binding molecule is characterized in that the first polypeptide comprises two external domains or fragments thereof of 4-1BBL linked to each other by a peptide linker, and the second polypeptide comprises one external domain or fragment thereof of 4-1BBL), (c) A half-life extension Fc domain comprising a first subunit and a second subunit capable of stable association as described herein, Includes, The Fc domain binding portion provides an immunoactivating fragment crystallizable (Fc) domain binding molecule that does not specifically bind to half-life extension Fc domains.

[0328] In yet another aspect, the present invention relates to an immunoactivating fragment crystallizable (Fc) domain binding molecule, (a) Two or more Fc domain binding moieties that specifically bind to the target Fc domain described herein, (b) A first and second polypeptide linked to each other by a disulfide bond (the immunoactivating Fc domain binding molecule is characterized in that the first polypeptide comprises two external domains or fragments thereof of 4-1BBL linked to each other by a peptide linker, and the second polypeptide comprises one external domain or fragment thereof of 4-1BBL), (c) A half-life extension Fc domain comprising a first subunit and a second subunit capable of stable association as described herein, Includes, The Fc domain binding portion provides an immunoactivating fragment crystallizable (Fc) domain binding molecule that does not specifically bind to half-life extension Fc domains.

[0329] In one embodiment, the present invention is an immunoactivating fragment crystallizable (Fc) domain binding molecule, (a) Two Fc domain binding moieties that specifically bind to the target Fc domain described herein, (b) A first and second polypeptide linked to each other by a disulfide bond (the antigen-binding molecule is characterized in that the first polypeptide comprises two external domains or fragments thereof of 4-1BBL linked to each other by a peptide linker, and the second polypeptide comprises one external domain or fragment thereof of 4-1BBL), (c) A half-life extension Fc domain comprising a first subunit and a second subunit capable of stable association as described herein, Includes, The Fc domain binding portion provides an immunoactivating fragment crystallizable (Fc) domain binding molecule that does not specifically bind to half-life extension Fc domains.

[0330] In a further embodiment, the present invention provides an immunoactivating fragment crystallizable (Fc) domain binding molecule, as previously defined herein, in which the Fc domain binding portion that specifically binds to a target Fc domain is selected from the group consisting of antibodies, antibody fragments, and scaffold antigen-binding proteins.

[0331] In one embodiment, an immunoactivating fragment crystallizable (Fc) domain binding molecule is provided herein, wherein the Fc domain binding portion that specifically binds to a target Fc domain is selected from the group consisting of an antibody fragment, a Fab molecule, a crossover Fab molecule, a single-stranded Fab molecule, an Fv molecule, an scFv molecule, a single-domain antibody, or aVH and a scaffold antigen-binding protein.

[0332] In one embodiment, the Fc domain binding portion that specifically binds to the target Fc domain is aVH or a scaffold antigen-binding protein.

[0333] In certain embodiments, an immunoactivating fragment crystallizable (Fc) domain binding molecule is provided, wherein the Fc domain binding portion that specifically binds to a target Fc domain is a Fab molecule or a crossover Fab molecule. In particular, the Fc domain binding portion that specifically binds to a target Fc domain is a Fab molecule.

[0334] In a further embodiment, an immunoactivating fragment crystallizable (Fc) domain binding molecule is provided according to the present invention, wherein a peptide comprising two external domains or fragments thereof of 4-1BBL linked to each other by a first peptide linker is fused at its C-terminus to the CH1 domain of a heavy chain by a second peptide linker, and one external domain or fragment thereof of 4-1BBL is fused at its C-terminus to the CL domain on a light chain by a third peptide linker.

[0335] In another embodiment, an immunoactivating fragment crystallizable (Fc) domain binding molecule is provided according to the present invention, wherein a peptide comprising two external domains or fragments thereof of 4-1BBL linked to each other by a first peptide linker is fused at its C-terminus to the CL domain of a heavy chain by a second peptide linker, and one external domain or fragment thereof of 4-1BBL is fused at its C-terminus to the CH1 domain on a light chain by a third peptide linker.

[0336] In a further embodiment, the present invention relates to an immunoactivating fragment crystallizable (Fc) domain binding molecule according to the present invention, wherein a peptide comprising two external domains or fragments thereof of 4-1BBL linked to each other by a first peptide linker is fused to the CL domain of the light chain at its C-terminus by a second peptide linker, and one external domain or fragment thereof of 4-1BBL is fused to the CH1 domain of the heavy chain at its C-terminus by a third peptide linker.

[0337] In certain embodiments, the present invention relates to an immunoactivating fragment crystallizable (Fc) domain binding molecule defined above, wherein the peptide linker is (G4S)2.

[0338] In another embodiment, the immunoactivating fragment crystallizable (Fc) domain binding molecule as previously defined herein comprises an Fc domain composed of first and second subunits capable of stable association.

[0339] In one embodiment, the immunoactivating fragment crystallizable (Fc) domain binding molecule is, (a) A first light chain containing the amino acid sequence of Sequence ID No. 10, (b) A second light chain containing the amino acid sequence of Sequence ID No. 129, (c) The first heavy chain containing the amino acid sequence of SEQ ID NO: 130, (d) Second heavy chain containing the amino acid sequence of SEQ ID NO: 131 and An immunoactivating fragment crystallizable (Fc) domain-binding molecule containing this domain is provided.

[0340] In one embodiment, the immunoactivating fragment crystallizable (Fc) domain binding molecule is, (a) A first light chain containing the amino acid sequence of SEQ ID NO: 15, (b) A second light chain containing the amino acid sequence of Sequence ID No. 129, (c) The first heavy chain containing the amino acid sequence of SEQ ID NO: 130, (d) Second heavy chain containing the amino acid sequence of SEQ ID NO: 132 An immunoactivating fragment crystallizable (Fc) domain-binding molecule containing this domain is provided.

[0341] In further embodiments, one of the above-described immunoactivating fragment crystallizable (Fc) domain binding molecules is provided, further comprising substitution at position P329 (numbered according to the Kabat EU index) with an amino acid selected from the list consisting of arginine (R), leucine (L), isoleucine (I), and alanine (A).

[0342] Immunoactivating Fc domain binding molecule containing the Fc receptor immune activation moiety In one embodiment of the present invention, the immunoactivating moiety is an Fc receptor. In one embodiment, the Fc receptor is an activated Fc receptor. In one embodiment, the immunoactivating moiety induces ACDD. In one embodiment, the Fc receptor is selected from a list consisting of FcγRIIIa (CD16a), FcγRI (CD64), FcγRIIa (CD32), and FcαRI (CD89). In a particular embodiment, the immunoactivating moiety is FcγRIIIa (CD16a) or a fragment thereof.

[0343] In certain embodiments, the immunoactivating portion is FcγRIIa(CD32) or a fragment thereof. In certain embodiments, the immunoactivating portion is FcαRI(CD89) or a fragment thereof.

[0344] Further specific immune-activating Fc domain binding molecules according to the present invention An immune-activating Fc domain binding molecule, (a) A Fab molecule, (i) Heavy chain variable region (VH), (a) Heavy chain complementarity determining region (CDR H) 1 amino acid sequence RYWMN (SEQ ID NO: 1); (b) A CDR H2 amino acid sequence selected from the group consisting of EITPDSSTINYTPSLKD (SEQ ID NO: 2), EITPDSSTINYTPSLKG (SEQ ID NO: 11), and EITPDSSTINYAPSLKG (SEQ ID NO: 16); and (c) CDR H3 amino acid sequence PYDYGAWFAS (SEQ ID NO: 3) The heavy chain variable region (VH), (ii) The light chain variable region (VL), (d) Light chain complementarity determination region (CDR L) 1 amino acid sequence RSSTGAVTTSNYAN (SEQ ID NO: 4); (e) CDR L2 amino acid sequence GTNKRAP (SEQ ID NO: 5); and (f) CDR L3 amino acid sequence ALWYSNHWV (SEQ ID NO: 6) A Fab molecule containing a light chain variable region (VL), (b) The IgG1 Fc domain including the substitution with arginine (R) at position P329 (numbering according to Kabat's EU index), (c) The immunoactivating portion described herein and An immune-activating Fc domain-binding molecule is provided, which includes this component.

[0345] An immune-activating Fc domain binding molecule, (a) A Fab molecule, (i) Heavy chain variable region (VH), (a) Heavy chain complementarity determining region (CDR H) 1 amino acid sequence RYWMN (SEQ ID NO: 1); (b) CDR H2 amino acid sequence EITPDSSTINYTPSLKD (SEQ ID NO: 2); and (c) CDR H3 amino acid sequence PYDYGAWFAS (SEQ ID NO: 3) The heavy chain variable region (VH), (ii) The light chain variable region (VL), (d) Light chain complementarity determination region (CDR L) 1 amino acid sequence RSSTGAVTTSNYAN (SEQ ID NO: 4); (e) CDR L2 amino acid sequence GTNKRAP (SEQ ID NO: 5); and (f) CDR L3 amino acid sequence ALWYSNHWV (SEQ ID NO: 6) A Fab molecule containing a light chain variable region (VL), (b) The IgG1 Fc domain including the substitution with arginine (R) at position P329 (numbering according to Kabat's EU index), (c) The immunoactivating portion described herein and An immune-activating Fc domain-binding molecule is provided, which includes this component.

[0346] An immune-activating Fc domain binding molecule, (a) A Fab molecule, (i) Heavy chain variable region (VH), (a) Heavy chain complementarity determining region (CDR H) 1 amino acid sequence RYWMN (SEQ ID NO: 1); (b) CDR H2 amino acid sequence EITPDSSTINYTPSLKG (SEQ ID NO: 11); and (c) CDR H3 amino acid sequence PYDYGAWFAS (SEQ ID NO: 3) The heavy chain variable region (VH), (ii) The light chain variable region (VL), (d) Light chain complementarity determination region (CDR L) 1 amino acid sequence RSSTGAVTTSNYAN (SEQ ID NO: 4); (e) CDR L2 amino acid sequence GTNKRAP (SEQ ID NO: 5); and (f) CDR L3 amino acid sequence ALWYSNHWV (SEQ ID NO: 6) A Fab molecule containing a light chain variable region (VL), (b) The IgG1 Fc domain including the substitution with arginine (R) at position P329 (numbering according to Kabat's EU index), (c) The immunoactivating portion described herein and An immune-activating Fc domain-binding molecule containing a Fab molecule is provided.

[0347] An immune-activating Fc domain binding molecule, (a) A Fab molecule, (i) Heavy chain variable region (VH), (a) Heavy chain complementarity determining region (CDR H) 1 amino acid sequence RYWMN (SEQ ID NO: 1); (b) CDR H2 amino acid sequence EITPDSSTINYAPSLKG (SEQ ID NO: 16); and (c) CDR H3 amino acid sequence PYDYGAWFAS (SEQ ID NO: 3) The heavy chain variable region (VH), (ii) The light chain variable region (VL), (d) Light chain complementarity determination region (CDR L) 1 amino acid sequence RSSTGAVTTSNYAN (SEQ ID NO: 4); (e) CDR L2 amino acid sequence GTNKRAP (SEQ ID NO: 5); and (f) CDR L3 amino acid sequence ALWYSNHWV (SEQ ID NO: 6) A Fab molecule containing a light chain variable region (VL), (b) The IgG1 Fc domain including the substitution with arginine (R) at position P329 (numbering according to Kabat's EU index), (c) The immunoactivating portion described herein and An immune-activating Fc domain-binding molecule containing a Fab molecule is provided.

[0348] targeting antibody The targeted antibody can bind to target cells (as shown in 43). As described above in this specification, the targeted antibody comprises a target Fc domain comprising at least one amino acid substitution of a first set. The targeted antibody may comprise any of the modifications and / or substitutions described above in this specification, in particular at least one amino acid substitution of a first set as described above in this specification. According to the concept of the present invention, as described above in this specification, the targeted antibody cross-links / links / connects the immunoactivating Fc domain binding molecule of the present invention to target cells (see, for example, Figures 1, 12, 13, 38, and 43).

[0349] In one embodiment, the present invention provides a targeted antibody that binds to a target antigen on a target cell. In one embodiment, an isolated targeted antibody that binds to a target antigen on a target cell is provided. In one embodiment, the present invention provides an antibody that specifically binds to an antigen selected from the list consisting of PD-L1, CD20, FolR1, CD25, FAP, EpCAM, STEAP1, Her2, and CEA.

[0350] In another embodiment, the targeted antibody can bind to immune cells, particularly T cells. In a preferred embodiment, the targeted antibody can bind to PD-1. Targeting PD-1 is particularly useful for targeting (delivering) cytokines to T cells. In one embodiment, the targeted antibody can bind to PD-1.

[0351] In a further embodiment, the targeted antibody described herein is of an IgG1 isotype / subclass.

[0352] In a further embodiment, the targeted antibody described herein comprises the heavy chain or constant portion thereof of SEQ ID NO: 146. In another embodiment, the antibody according to any of the above embodiments comprises the light chain or constant portion thereof of SEQ ID NO: 147.

[0353] In a further embodiment, the targeted antibody described herein comprises the heavy chain or constant portion thereof of SEQ ID NO: 148. In another embodiment, the antibody according to any of the above embodiments comprises the light chain or constant portion thereof of SEQ ID NO: 149.

[0354] In a further embodiment, the targeted antibody described herein comprises the heavy chain or constant portion thereof of SEQ ID NO: 150. In another embodiment, the antibody according to any of the above embodiments comprises the light chain or constant portion thereof of SEQ ID NO: 151.

[0355] In a further embodiment, the targeted antibody described herein comprises the heavy chain or constant portion thereof of SEQ ID NO: 152. In another embodiment, the antibody according to any of the above embodiments comprises the light chain or constant portion thereof of SEQ ID NO: 153.

[0356] In a further embodiment, the targeted antibody described herein comprises the heavy chain or constant portion thereof of SEQ ID NO: 154. In another embodiment, the antibody according to any of the above embodiments comprises the light chain or constant portion thereof of SEQ ID NO: 155.

[0357] In a further embodiment, the targeted antibody described herein comprises the heavy chain or constant portion thereof of SEQ ID NO: 156. In another embodiment, the antibody according to any of the above embodiments comprises the light chain or constant portion thereof of SEQ ID NO: 157.

[0358] In a further embodiment, the targeted antibody described herein comprises the heavy chain or constant portion thereof of SEQ ID NO: 158. In another embodiment, the antibody according to any of the above embodiments comprises the light chain or constant portion thereof of SEQ ID NO: 159.

[0359] In a further embodiment, the targeted antibody described herein comprises the heavy chain or constant portion thereof of SEQ ID NO: 160. In another embodiment, the antibody according to any of the above embodiments comprises the light chain or constant portion thereof of SEQ ID NO: 161.

[0360] In a further embodiment, the targeted antibody described herein comprises the heavy chain or constant portion thereof of SEQ ID NO: 162. In another embodiment, the antibody according to any of the above embodiments comprises the light chain or constant portion thereof of SEQ ID NO: 163.

[0361] In a further embodiment, the targeted antibody described herein comprises the heavy chain or constant portion thereof of SEQ ID NO: 164. In another embodiment, the antibody according to any of the above embodiments comprises the light chain or constant portion thereof of SEQ ID NO: 165.

[0362] In one embodiment, C-terminal glycine (Gly446) is further present in the heavy chain sequence described above. In another embodiment, C-terminal glycine (Gly446) and C-terminal lysine (Lys447) are further present.

[0363] Polynucleotides The present invention further provides isolated polynucleotides encoding immunoactivating Fc domain-binding molecules or fragments thereof as described herein. In some embodiments, the fragment is an antigen-binding fragment.

[0364] The polynucleotide encoding the immunoactivating Fc domain binding molecule of the present invention may be expressed as a single polynucleotide encoding the entire immunoactivating Fc domain binding molecule, or as multiple (e.g., two or more) polynucleotides co-expressed. Polypeptides encoded by co-expressed polynucleotides may associate, for example, by disulfide bonds or by other means, to form a functional immunoactivating Fc domain binding molecule. For example, the light chain portion of a Fab molecule may be encoded by a polynucleotide separate from the portion of the immunoactivating Fc domain binding molecule that includes the heavy chain portion of the Fab molecule, the Fc domain subunits, and optionally part of another Fab molecule. When co-expressed, the heavy chain polypeptide associates with the light chain polypeptide to form the Fab molecule. In another example, the portion of the immunoactivating Fc domain binding molecule that includes one of two Fc domain subunits and optionally part of one or more Fab molecules may be encoded by a polynucleotide separate from the portion of the immunoactivating Fc domain binding molecule that includes the other of the two Fc domain subunits and optionally part of another Fab molecule. When co-expressed, the Fc domain subunits associate to form the Fc domain.

[0365] In some embodiments, the isolated polynucleotide encodes the entire immunoactivating Fc domain-binding molecule according to the present invention as described herein. In other embodiments, the isolated polynucleotide encodes the polypeptide contained in the immunoactivating Fc domain-binding molecule according to the present invention as described herein.

[0366] In certain embodiments, the polynucleotide or nucleic acid is DNA. In other embodiments, the polynucleotide of the present invention is RNA, for example, in the form of messenger RNA (mRNA). The RNA of the present invention may be single-stranded or double-stranded.

[0367] Recombination method The immunoactivating Fc domain binding molecule of the present invention may be obtained, for example, by solid-state peptide synthesis (e.g., Merrifield solid-phase synthesis) or recombinant production. For recombinant production, one or more polynucleotides encoding the immunoactivating Fc domain binding molecule (fragment) are isolated, for example, as described above, and inserted into one or more vectors for further cloning and / or expression in host cells. Such polynucleotides can be readily isolated and sequenced using common procedures. In one embodiment, a vector, preferably an expression vector, containing one or more polynucleotides of the present invention is provided. An expression vector containing the coding sequence of the immunoactivating Fc domain binding molecule (fragment) can be constructed using methods well known to those skilled in the art, following appropriate transcription / translation control signals. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo recombination / genetic recombination. See, for example, the techniques described in Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, NY (1989); and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley Interscience, NY (1989). The expression vector may be a plasmid, part of a virus, or a nucleic acid fragment. The expression vector comprises an expression cassette in which a polynucleotide encoding an immunoactivating Fc domain-binding molecule (fragment) (i.e., coding region) is cloned in conjunction with a promoter and / or other transcriptional or translational regulatory elements. As used herein, “coding region” is a part of a nucleic acid consisting of codons that are translated into amino acids. “Stop codons” (TAG, TGA, or TAA) are not translated into amino acids but are considered part of the coding region (if present). However, any adjacent sequences such as promoters, ribosome-binding sites, transcriptional terminators, introns, and 5' and 3' untranslated regions are not part of the coding region.Two or more coding regions may be present in a single polynucleotide construct, for example, on a single vector, or in separate polynucleotide constructs, for example, on separate (different) vectors. Furthermore, any vector may contain a single coding region or two or more coding regions. For example, the vector of the present invention may encode one or more polypeptides that are separated into final proteins post-translation or concurrently with translation by proteolytic cleavage. In addition, the vector, polynucleotide, or nucleic acid of the present invention may encode heterologous coding regions that are fused to or not fused to the polynucleotide, or its variant or derivative, which encodes the immunoactivating Fc domain binding molecule (fragment) of the present invention. Heterologous coding regions include, but are not limited to, special elements or motifs such as secretory signaling peptides or heterologous functional domains. A responsive binding is when the coding region of a gene product, such as a polypeptide, is bound to one or more regulatory sequences such that the expression of the gene product is under the influence or control of the regulatory sequences. Two DNA fragments (such as a polypeptide coding region and a promoter bound to it) are "operably bound" if the induction of promoter function results in the transcription of mRNA encoding a desired gene product, and the nature of the linkage between the two DNA fragments does not interfere with the ability of an expression regulatory sequence that directs the expression of the gene product or the ability of the DNA template to be transcribed. Therefore, a promoter region can be said to be operably bound to a polypeptide-encoding nucleic acid if the promoter can result in the transcription of that nucleic acid. The promoter may be a cell-specific promoter that directs substantial transcription of DNA only in a given cell. In addition to promoters, other transcriptional regulatory elements, such as enhancers, operators, repressors, and transcription termination signals, can operably bind to polynucleotides that direct cell-specific transcription. Suitable promoters and other transcriptional regulatory regions are disclosed herein. Various transcriptional regulatory regions are known to those skilled in the art.These include, but are not limited to, transcriptional regulatory regions that function in vertebrate cells, such as, but are not limited to, cytomegalovirus-derived promoter and enhancer segments (e.g., early promoter, in combination with intron-A), Simianvirus 40 (e.g., early promoter), and retroviruses (e.g., Roussarcoma virus). Other transcriptional regulatory regions include those derived from vertebrate genes, such as actin, heat shock proteins, bovine growth hormone, and rabbit α-globin, and other sequences capable of regulating gene expression in eukaryotic cells. Further suitable transcriptional regulatory regions include tissue-specific promoters and enhancers, and induced promoters (e.g., promoter-induced tetracycline). Similarly, various translational regulatory elements are known to those skilled in the art. These include, but are not limited to, ribosome-binding sites, translation start and stop codons, and viral-derived elements (in particular, intrasequence ribosome entry sites, i.e., IRES, also known as CITE sequences). Furthermore, the expression cassette may also include other features such as chromosomal integration elements, including, for example, the origin of replication and / or the long terminal repeat sequence (LTR) of a retrovirus or the inverted terminal sequence (ITR) of an adeno-associated virus (AAV).

[0368] The polynucleotide and nucleic acid coding regions of the present invention can be coupled with further coding regions encoding secretory or signal peptides that direct the secretion of polypeptides encoded by the polynucleotide of the present invention. For example, if the secretion of an immunoactivating Fc domain binding molecule is desired, the DNA encoding the signal sequence can be placed upstream of the nucleic acid encoding the immunoactivating Fc domain binding molecule or a fragment thereof of the present invention. According to the signaling hypothesis, proteins secreted by mammalian cells have a signal peptide or secretory leader sequence that is cleaved from the mature protein when efflux transport across the rough endoplasmic reticulum of the growing protein chain is initiated. Those skilled in the art know that polypeptides secreted by vertebrate cells generally have a signal peptide fused to the N-terminus of the polypeptide, which is cleaved from the translated polypeptide to produce a secretory or “mature” form of polypeptide. In certain embodiments, native signal peptides, such as immunoglobulin heavy chain or light chain signal peptides, are used, or functional derivatives of sequences that retain the ability to rectify polypeptide fission are used. Alternatively, heterologous mammalian signal peptides or functional derivatives thereof may be used. For example, the wild-type leader sequence may be replaced with the leader sequence of human tissue plasminogen activator (TPA) or mouse β-glucuronidase.

[0369] DNA encoding a short protein sequence (e.g., a histidine tag) that can be used to facilitate subsequent purification or to label the immunoactivating Fc domain binding molecule may be included in or at the end of the immunoactivating Fc domain binding molecule (fragment) encoding a polynucleotide.

[0370] In further embodiments, host cells comprising one or more polynucleotides of the present invention are provided. In specific embodiments, host cells comprising one or more vectors of the present invention are provided. The polynucleotides and vectors may each incorporate, individually or in combination, any of the features described herein in relation to the polynucleotides and vectors. In one such embodiment, the host cell comprises a vector comprising a polynucleotide encoding (or part thereof) the immunoactivating Fc domain binding molecule of the present invention (e.g., transformed by or transfected by therewith). As used herein, the term “host cell” refers to any type of cell line capable of being manipulated to produce the immunoactivating Fc domain binding molecule or fragments thereof of the present invention. Host cells suitable for replicating and assisting the expression of the immunoactivating Fc domain binding molecule are well known in the Art. Such cells may, where appropriate, be transfected or transduced with a particular expression vector to grow cells containing large quantities of vector for inoculation into a large-scale fermenter, thereby obtaining sufficient quantities of the immunoactivating Fc domain binding molecule for clinical use. Suitable host cells include prokaryotic microorganisms (e.g., Escherichia coli) or various eukaryotic cells, such as Chinese hamster ovary cells (CHO) and insect cells. For example, polypeptides may be produced within bacteria if glycosylation is not particularly required. After expression, polypeptides may be isolated from bacterial cell paste in a suitable fraction and further purified. In addition to prokaryotes, eukaryotic microorganisms, such as filamentous fungi or yeasts, are suitable hosts for cloning or expression of polypeptide-encoding vectors, including fungal and yeast strains with "humanized" glycosylation pathways that produce polypeptides with partially or completely human glycosylation patterns. See Gerngross, Nat Biotech 22, 1409-1414 (2004) and Li et al., Nat Biotech 24, 210-215 (2006). Suitable host cells for (glycosylated) polypeptide expression can also be obtained from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant cells and insect cells.Numerous baculovirus strains have been identified and can be used in combination with insect cells, particularly for transfection of Spodoptera frugiperda cells. Plant cell cultures can also be used as hosts. See, for example, U.S. Patents 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (which describe PLANTIBODIES® technology for antibody production in transgenic plants). Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted for growth in suspension may be useful. Other examples of useful mammalian host cell lines include the CV1 monkey kidney cell line transformed by SV40 (COS-7); human embryonic kidney cells (e.g., 293 or 293T cells described in Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK); mouse Sertoli cells (e.g., TM4 cells described in Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical tumor cells (HELA); canine kidney cells (MDCK); buffalo rat hepatocytes (BRL 3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary tumor cells (MMT 060562); e.g., Mather et al., Annals These include TRI cells; MRC 5 cells; and FS4 cells, as described in NYAcad.Sci.383:44-68(1982). Other useful mammalian host cell lines include dhfr. -Examples of suitable host cells include Chinese hamster ovary (CHO) cells (Urlaub et al., Proc Natl Acad Sci USA 77, 4216 (1980)), myeloma cell lines such as YO, NS0, P3X63, and Sp2 / 0. For a review of specific mammalian host cells suitable for protein production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKCLo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003). Host cells include cultured cells, such as mammalian cultured cells, yeast cells, insect cells, bacterial cells, and plant cells, but also cells contained in transgenic animals, transgenic plants, or cultured plants or animal tissues. In one embodiment, the host cell is a eukaryotic cell, preferably a mammalian cell, such as a Chinese hamster ovary (CHO) cell, a human embryonic kidney (HEK) cell, or a lymphocyte (e.g., Y0, NS0, Sp20 cells).

[0371] Standard techniques for expressing foreign genes in these systems are well known in the art. By manipulating cells that express polypeptides containing either the heavy or light chain of the antigen-binding domain, such as antibodies, it is possible to also express the other antibody chain, so that the expressed product becomes an antibody possessing both heavy and light chains.

[0372] In one embodiment, a method for producing an immune-activating Fc domain binding molecule according to the present invention is provided, which comprises culturing host cells containing a polynucleotide encoding the immune-activating Fc domain binding molecule provided herein under conditions suitable for the expression of the immune-activating Fc domain binding molecule, and optionally...

Claims

1. An immunoactivating fragment crystallizable (Fc) domain binding molecule, (a) an Fc domain binding moiety that specifically binds to a target Fc domain containing at least one amino acid substitution of the first set, (b) Immune activation portion and (c) Half-life extension Fc domain and Includes, The Fc domain binding portion is specifically capable of binding to an IgG1 Fc domain containing the amino acid substitution P329G (numbering according to Kabat's EU index), The half-life extension Fc domain comprises at least one amino acid substitution of the second set, At least one amino acid substitution in the second set includes a substitution at position P329 (numbered according to Kabat's EU index) by an amino acid selected from the list consisting of arginine (R), leucine (L), isoleucine (I), and alanine (A), An immune-activating Fc domain binding molecule.

2. The immunoactivating Fc domain binding molecule according to Claim 1, wherein the Fc domain binding portion does not specifically bind to the half-life extension Fc domain.

3. The immunoactivating Fc domain binding molecule according to claim 1 or 2, wherein at least one amino acid substitution in the first set reduces the binding affinity and / or effector function to the Fc receptor, and at least one amino acid substitution in the second set includes one or more amino acid substitutions at the same amino acid position as at least one amino acid substitution in the first set, and the amino acids of at least one amino acid substitution in the second set are substituted with different amino acids at the same position as at least one amino acid substitution in the first set.

4. The immunoactivating Fc domain binding molecule according to any one of claims 1 to 3, wherein at least one amino acid substitution of the first set comprises at least one amino acid substitution at a position selected from the list consisting of 233, 234, 235, 238, 253, 265, 269, 270, 297, 310, 331, 327 and 435 (numbering according to Kabat's EU index).

5. The immunoactivating Fc domain binding molecule according to any one of claims 1 to 4, wherein at least one amino acid substitution of the second set comprises at least one amino acid substitution at a position selected from the list consisting of 233, 234, 235, 238, 253, 265, 269, 270, 297, 310, 331, 327 and 435 (numbering according to Kabat's EU index).

6. The immunoactivating Fc domain binding molecule according to any one of claims 1 to 5, wherein at least one amino acid substitution of the second set comprises a substitution by arginine (R) at position P329 (numbered according to Kabat's EU index).

7. The immunoactivating Fc domain binding molecule according to any one of claims 1 to 6, wherein the Fc domain binding portion is capable of specific binding to an IgG1 Fc domain containing the amino acid substitution P329G (numbering according to Kabat's EU index), but is not capable of specific binding to a parental non-mutant IgG1 Fc domain.

8. The immunoactivating Fc domain binding molecule according to any one of claims 1 to 7, wherein at least one amino acid substitution of the first set comprises at least one amino acid substitution at a position selected from the group L234, L235 (Kabat's EU index numbering).

9. The immunoactivating Fc domain binding molecule according to any one of claims 1 to 8, wherein at least one amino acid substitution of the second set comprises at least one amino acid substitution at a position selected from the group L234, L235 (Kabat's EU index numbering).

10. The immunoactivating Fc domain binding molecule according to any one of claims 1 to 6, wherein the target Fc domain comprises three amino acid substitutions that reduce binding to and / or effector function of the activated Fc receptor, the amino acid substitutions being L234A, L235A, and P329G (Kabat's EU index numbering).

11. The immunoactivating Fc domain binding molecule according to any one of claims 1 to 10, wherein the half-life extension Fc domain comprises three amino acid substitutions that reduce binding to and / or effector function of the activated Fc receptor, the amino acid substitutions being L234A, L235A, and P329X (Kabat's EU index numbering), where X is an amino acid other than glycine (G).

12. The immunoactivating Fc domain binding molecule according to any one of claims 1 to 11, wherein the Fc domain binding portion and / or the immunoactivating portion is a Fab molecule.

13. The Fc domain binding portion is capable of specific binding to an IgG1 Fc domain containing the amino acid substitution P329G (numbering according to Kabat's EU index), and the Fc domain binding portion, (i) Heavy chain variable region (VH), (a) Heavy chain complementarity determining region (CDR H) 1 amino acid sequence RYWMN (SEQ ID NO: 1); (b) CDR H2 amino acid sequences selected from the group consisting of EITPDSSTINYTPSLKD (SEQ ID NO: 2), EITPDSSTINYTPSLKG (SEQ ID NO: 11), and EITPDSSTINYAPSLKG (SEQ ID NO: 16); and (c) CDR H3 amino acid sequence PYDYGAWFAS (SEQ ID NO: 3) The heavy chain variable region (VH), (ii) Light chain variable region (VL), (d) Light chain complementarity determining region (CDR L) 1 amino acid sequence RSSTGAVTTTSNYAN (SEQ ID NO: 4); (e) CDR L2 amino acid sequence GTNKRAP (SEQ ID NO: 5); and (f) CDR L3 amino acid sequence ALWYSNHWV (SEQ ID NO: 6) Including the light chain variable region (VL) and An immunoactivating Fc domain binding molecule according to any one of claims 1 to 12, comprising:

14. The Fc domain binding portion is capable of specific binding to an IgG1 Fc domain containing the amino acid substitution P329G (numbering according to Kabat's EU index), and the Fc domain binding portion, (i) Heavy chain variable region (VH), (a) Heavy chain complementarity determining region (CDR H) 1 amino acid sequence RYWMN (SEQ ID NO: 1); (b) CDR H2 amino acid sequence EITPDSSTINYTPSLKD (SEQ ID NO: 2); and (c) CDR H3 amino acid sequence PYDYGAWFAS (SEQ ID NO: 3) The heavy chain variable region (VH), (ii) Light chain variable region (VL), (d) Light chain complementarity determining region (CDR L) 1 amino acid sequence RSSTGAVTTTSNYAN (SEQ ID NO: 4); (e) CDR L2 amino acid sequence GTNKRAP (SEQ ID NO: 5); and (f) CDR L3 amino acid sequence ALWYSNHWV (SEQ ID NO: 6) Including the light chain variable region (VL) and The immunoactivating Fc domain binding molecule according to claim 13, comprising:

15. The Fc domain binding portion is capable of specific binding to an IgG1 Fc domain containing the amino acid substitution P329G (numbering according to Kabat's EU index), and the Fc domain binding portion, (i) Heavy chain variable region (VH), (a) Heavy chain complementarity determining region (CDR H) 1 amino acid sequence RYWMN (SEQ ID NO: 1); (b) CDR H2 amino acid sequence EITPDSSTINYTPSLKG (SEQ ID NO: 11); and (c) CDR H3 amino acid sequence PYDYGAWFAS (SEQ ID NO: 3) The heavy chain variable region (VH), (ii) Light chain variable region (VL), (d) Light chain complementarity determining region (CDR L) 1 amino acid sequence RSSTGAVTTTSNYAN (SEQ ID NO: 4); (e) CDR L2 amino acid sequence GTNKRAP (SEQ ID NO: 5); and (f) CDR L3 amino acid sequence ALWYSNHWV (SEQ ID NO: 6) Including the light chain variable region (VL) and The immunoactivating Fc domain binding molecule according to claim 13, comprising:

16. The Fc domain binding portion is capable of specific binding to an IgG1 Fc domain containing the amino acid substitution P329G (numbering according to Kabat's EU index), and the Fc domain binding portion, (i) Heavy chain variable region (VH), (a) Heavy chain complementarity determining region (CDR H) 1 amino acid sequence RYWMN (SEQ ID NO: 1); (b) CDR H2 amino acid sequence EITPDSSTINYAPSLKG (SEQ ID NO: 16); and (c) CDR H3 amino acid sequence PYDYGAWFAS (SEQ ID NO: 3) The heavy chain variable region (VH), (ii) Light chain variable region (VL), (d) Light chain complementarity determining region (CDR L) 1 amino acid sequence RSSTGAVTTTSNYAN (SEQ ID NO: 4); (e) CDR L2 amino acid sequence GTNKRAP (SEQ ID NO: 5); and (f) CDR L3 amino acid sequence ALWYSNHWV (SEQ ID NO: 6) Including the light chain variable region (VL) and The immunoactivating Fc domain binding molecule according to claim 13, comprising:

17. The immunoactivating Fc domain binding molecule according to any one of claims 1 to 16, wherein the Fc domain binding portion is capable of specific binding to an IgG1 Fc domain containing an amino acid substitution P329G (numbering according to Kabat's EU index), and the Fc domain binding portion comprises a heavy chain variable region sequence which is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs. 7, SEQ ID NOs. 12, SEQ ID NOs. 17, and SEQ ID NOs. 19, and a light chain variable region sequence which is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs. 8 and SEQ ID NOs.

13.

18. The Fc domain binding portion is capable of specific binding to an IgG1 Fc domain containing the amino acid substitution P329G (numbering according to Kabat's EU index), and the Fc domain binding portion, (i) A heavy chain variable region sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to sequence number 7, and a light chain variable region sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to sequence number 8, (ii) A heavy chain variable region sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to sequence number 12, and a light chain variable region sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to sequence number 13, (iii) A heavy chain variable region sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 17, and a light chain variable region sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 13, or (iv) A heavy chain variable region sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 19, and a light chain variable region sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:

13. The immunoactivating Fc domain binding molecule according to claim 17, comprising:

19. The immunoactivating Fc domain binding molecule according to claim 18, wherein the Fc domain binding portion is capable of specific binding to an IgG1 Fc domain containing an amino acid substitution P329G (numbering according to Kabat's EU index), and the Fc domain binding portion includes the heavy chain variable region sequence of SEQ ID NO: 19 and the light chain variable region of SEQ ID NO:

13.

20. The immunoactivating Fc domain binding molecule according to any one of claims 1 to 19, wherein the Fc domain binding portion comprises a first Fab molecule and the immunoactivating portion comprises a second Fab molecule.

21. d) The immunoactivating Fc domain binding molecule according to claim 20, further comprising a third Fab molecule that specifically binds to the target Fc domain comprising at least one amino acid substitution of the first set.

22. The immunoactivating Fc domain binding molecule according to claim 21, wherein the third Fab molecule is identical to the first Fab molecule.

23. The immunoactivating Fc domain binding molecule according to any one of claims 1 to 22, wherein the immunoactivating portion is capable of specific binding to activated T cell antigens.

24. The immunoactivating Fc domain binding molecule according to claim 23, wherein the activated T cell antigen is CD3.

25. The immunoactivating Fc domain binding molecule according to claim 23 or 24, wherein the activated T cell antigen is CD3 epsilon.

26. The immunoactivating Fc domain binding molecule according to any one of claims 23 to 25, wherein the immunoactivating portion specifically binds to an activated T cell antigen.

27. The immunoactivating Fc domain binding molecule according to any one of claims 23 to 26, wherein the immunoactivating portion is a Fab molecule.

28. The immunoactivating Fc domain binding molecule according to claim 27, wherein the activated T cell antigen is CD3, and the Fab molecule that specifically binds to the activated T cell antigen includes heavy chain complementarity determining region (CDR) 1 of SEQ ID NO: 35, heavy chain CDR 2 of SEQ ID NO: 37, heavy chain CDR 3 of SEQ ID NO: 43, light chain CDR 1 of SEQ ID NO: 53, light chain CDR 2 of SEQ ID NO: 54, and light chain CDR 3 of SEQ ID NO:

55.

29. The immunoactivating Fc domain binding molecule according to claim 28, wherein the activated T cell antigen is CD3, and the Fab molecule that specifically binds to the activated T cell antigen comprises a heavy chain variable region containing an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 49, and a light chain variable region containing an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:

56.

30. The immunoactivating Fc domain binding molecule according to claim 27, wherein the activated T cell antigen is CD3, and the Fab molecule that specifically binds to the activated T cell antigen comprises the heavy chain complementarity determining region (CDR) 1 of SEQ ID NO: 35, the heavy chain CDR 2 of SEQ ID NO: 33, the heavy chain CDR 3 of SEQ ID NO: 176, the light chain CDR 1 of SEQ ID NO: 53, the light chain CDR 2 of SEQ ID NO: 54, and the light chain CDR 3 of SEQ ID NO:

55.

31. The immunoactivating Fc domain binding molecule according to claim 30, wherein the activated T cell antigen is CD3, and the Fab molecule that specifically binds to the activated T cell antigen comprises a heavy chain variable region containing an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 177, and a light chain variable region containing an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:

56.

32. The immunoactivating Fc domain binding molecule according to claim 27, wherein the activated T cell antigen is CD3, and the Fab molecule that specifically binds to the activated T cell antigen includes heavy chain complementarity determining region (CDR) 1 of SEQ ID NO: 34, heavy chain CDR 2 of SEQ ID NO: 37, heavy chain CDR 3 of SEQ ID NO: 41, light chain CDR 1 of SEQ ID NO: 53, light chain CDR 2 of SEQ ID NO: 54, and light chain CDR 3 of SEQ ID NO:

55.

33. The immunoactivating Fc domain binding molecule according to claim 32, wherein the activated T cell antigen is CD3, and the Fab molecule that specifically binds to the activated T cell antigen comprises a heavy chain variable region containing an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 47, and a light chain variable region containing an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:

56.

34. An immunoactivating fragment crystallizable (Fc) domain binding molecule that is specifically capable of binding to an IgG1 Fc domain containing an amino acid substitution P329G and CD3, (a) A first light chain comprising an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to Sequence ID No. 89, (b) A second light chain comprising an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 70, (c) A first heavy chain comprising an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 178, and (d) A second heavy chain having an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:

179. An immune-activating Fc domain-binding molecule containing this molecule.

35. An immunoactivating fragment crystallizable (Fc) domain binding molecule that is specifically capable of binding to an IgG1 Fc domain containing an amino acid substitution P329G and CD3, (a) A first light chain comprising an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to Sequence ID No. 89, (b) A second light chain comprising an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 68, (c) A first heavy chain comprising an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 178, and (d) A second heavy chain having an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:

179. An immune-activating Fc domain-binding molecule containing this molecule.

36. An immunoactivating fragment crystallizable (Fc) domain binding molecule that is specifically capable of binding to an IgG1 Fc domain containing an amino acid substitution P329G and CD3, (a) A first light chain comprising an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to Sequence ID No. 89, (b) A second light chain comprising an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 180, (c) A first heavy chain comprising an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 178, and (d) A second heavy chain having an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:

179. An immune-activating Fc domain-binding molecule containing this molecule.

37. The immunoactivating Fc domain binding molecule according to any one of claims 1 to 22, wherein the immunoactivating portion is capable of specific binding to a costimulatory T cell antigen.

38. The immunoactivating Fc domain binding molecule according to claim 37, wherein the aforementioned costimulatory T cell antigen is CD28.

39. The immunoactivating Fc domain binding molecule according to claim 37 or 38, wherein the immunoactivating portion specifically binds to a costimulatory T cell antigen.

40. The immunoactivating Fc domain binding molecule according to any one of claims 37 to 39, wherein the immunoactivating portion specifically binds to CD28.

41. The immunoactivating Fc domain binding molecule according to any one of claims 37 to 40, wherein the immunoactivating portion is a Fab molecule.

42. The immunoactivating Fc domain binding molecule according to claim 41, wherein the Fab molecule that specifically binds to CD28 includes the heavy chain complementarity determining region (CDR) 1 of SEQ ID NO: 94, the heavy chain CDR 2 of SEQ ID NO: 95, the heavy chain CDR 3 of SEQ ID NO: 96, the light chain CDR 1 of SEQ ID NO: 97, the light chain CDR 2 of SEQ ID NO: 98, and the light chain CDR 3 of SEQ ID NO: 99, or the heavy chain complementarity determining region (CDR) 1 of SEQ ID NO: 94, the heavy chain CDR 2 of SEQ ID NO: 95, the heavy chain CDR 3 of SEQ ID NO: 102, the light chain CDR 1 of SEQ ID NO: 103, the light chain CDR 2 of SEQ ID NO: 98, and the light chain CDR 3 of SEQ ID NO:

99.

43. The immunoactivating Fc domain binding molecule according to claim 42, wherein the Fab molecule that specifically binds to CD28 comprises a heavy chain variable region containing an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 100; a light chain variable region containing an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 101 or the amino acid sequence of SEQ ID NO: 104; and a light chain variable region containing an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:

105.

44. The immunoactivating Fc domain binding molecule according to any one of claims 1 to 22, wherein the immunoactivating portion is capable of specific binding to a costimulatory T cell antigen.

45. The immunoactivating Fc domain binding molecule according to claim 44, wherein the aforementioned co-stimulatory T cell antigen is 4-1BB.

46. The immunoactivating Fc domain binding molecule according to claim 44 or 45, wherein the immunoactivating portion specifically binds to a costimulatory T cell antigen.

47. The immunoactivating Fc domain binding molecule according to any one of claims 44 to 46, wherein the immunoactivating portion specifically binds to 4-1BB.

48. The immunoactivating Fc domain binding molecule according to any one of claims 44 to 47, wherein the immunoactivating portion is a Fab molecule.

49. The immunoactivating Fc domain binding molecule according to claim 48, wherein the Fab molecule that specifically binds to 4-1BB includes heavy chain complementarity determining region (CDR) 1 of SEQ ID NO: 133, heavy chain CDR 2 of SEQ ID NO: 134, heavy chain CDR 3 of SEQ ID NO: 135, light chain CDR 1 of SEQ ID NO: 136, light chain CDR 2 of SEQ ID NO: 137, and light chain CDR 3 of SEQ ID NO:

138.

50. The immunoactivating Fc domain binding molecule according to claim 49, wherein the Fab molecule that specifically binds to 4-1BB comprises i) a heavy chain variable region containing an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 139, and a light chain variable region containing an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:

140.

51. The immunoactivating fragment crystallizable (Fc) domain binding molecule according to any one of claims 1 to 19, wherein the immunoactivating portion is a cytokine.

52. The immunoactivating fragment crystallizable (Fc) domain binding molecule according to claim 51, wherein the cytokine is selected from the group consisting of IL2, IL7, IL15, IL18, IFNa, and IFNg.

53. The immunoactivating fragment crystallizable (Fc) domain-binding molecule according to claim 51 or 52, wherein the immunoactivating portion is a mutant interleukin-2 (IL-2) polypeptide.

54. The immunoactivating fragment crystallizable (Fc) domain binding molecule according to claim 53, wherein the mutant IL-2 polypeptide is a human IL-2 molecule comprising amino acid substitutions F42A, Y45A and L72G (numbering for SEQ ID NO: 166, which is a human IL-2 sequence).

55. The immunoactivating fragment crystallizable (Fc) domain binding molecule according to claim 54, wherein the mutant IL-2 polypeptide further comprises amino acid substitution T3A and / or amino acid substitution C125A.

56. The immunoactivating fragment crystallizable (Fc) domain binding molecule according to any one of claims 53 to 55, wherein the mutant IL-2 polypeptide comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 167, and the mutant IL-2 polypeptide exhibits a reduced affinity for high-affinity IL-2 receptors and substantially similar affinity for medium-affinity IL-2 receptors, respectively, compared to the wild-type IL-2 polypeptide.

57. The immunoactivating fragment crystallizable (Fc) domain binding molecule according to any one of claims 53 to 56, wherein the mutant IL-2 polypeptide comprises the amino acid sequence of SEQ ID NO:

167.

58. The immunoactivating Fc domain binding molecule according to any one of claims 1 to 19, wherein the immunoactivating portion comprises three external domains of 4-1BBL or a fragment thereof.

59. The immunoactivating Fc domain binding molecule according to any one of claims 1 to 19, wherein the immunoactivating portion comprises a first polypeptide and a second polypeptide, the first polypeptide comprising a first heavy chain constant (CH1) domain or a light chain constant (CL) domain, the second polypeptide comprising a CL domain or a CH1 domain, the second polypeptide linked to the first polypeptide by a disulfide bond between the CH1 domain and the CL domain, the first polypeptide comprising two external domains or fragments thereof of 4-1BBL connected to each other and to the CH1 domain or the CL domain by a peptide linker, and the second polypeptide comprising one external domain or fragment thereof of 4-1BBL connected to the CL domain or the CH1 domain of the polypeptide via a peptide linker.

60. The immunoactivating Fc domain binding molecule according to claim 58 or 59, wherein the external domain or fragment thereof of 4-1BBL comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 117, 118, 119, 120, 121, 122, 123, and 124.

61. The immunoactivating Fc domain binding molecule according to any one of claims 58 to 60, wherein the immunoactivating portion comprises a first polypeptide and a second polypeptide linked to each other by a disulfide bond, and the antigen-binding molecule is characterized in that the first polypeptide contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 125, SEQ ID NOs: 126, SEQ ID NOs: 127 and SEQ ID NOs: 128, and the second polypeptide contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 117, SEQ ID NOs: 121, SEQ ID NOs: 119 and SEQ ID NOs:

120.

62. An immunoactivating Fc domain binding molecule according to any one of claims 1 to 19 or 58 to 61, comprising a first heavy chain and a first light chain including the Fc domain binding portion, and a second heavy chain and a second light chain including the immunoactivating portion, wherein the second heavy chain comprises the first polypeptide comprising two external domains or fragments thereof of 4-1BBL connected to each other and to the CH1 domain or the CL domain by a peptide, and the second light chain comprises the second polypeptide comprising one external domain or fragment thereof of 4-1BBL connected to the CL domain or the CH1 domain of the polypeptide via a peptide linker.

63. An immunoactivating Fc domain binding molecule according to any one of claims 59 to 62, wherein the first peptide, comprising two external domains or fragments thereof of 4-1BBL linked to each other by a first peptide linker, is fused at its C-terminus to a CL domain which is part of a heavy chain by a second peptide linker, and the second peptide, comprising one external domain or fragment thereof of 4-1BBL, is fused at its C-terminus to a CH1 domain which is part of a light chain by a third peptide linker.

64. The immunoactivating Fc domain binding molecule according to any one of claims 1 to 19, wherein the immunoactivating portion is capable of specific binding to an Fc receptor.

65. The immunoactivating Fc domain binding molecule according to claim 64, wherein the Fc receptor is an Fc gamma receptor.

66. The immunoactivating Fc domain binding molecule according to claim 64 or 65, wherein the Fc receptor is CD16.

67. One or more isolated polynucleotides encoding an immunoactivating Fc domain binding molecule according to any one of claims 1 to 66.

68. One or more vectors comprising the polynucleotide(s) described in claim 67.

69. A host cell comprising the polynucleotide(s) described in claim 67 or the vector(s) described in claim 68.

70. A method for producing an immunoactivating fragment crystallizable (Fc) domain-binding molecule, comprising: a) culturing the host cells described in claim 69 under conditions suitable for the expression of the immunoactivating Fc domain-binding molecule; and b) recovering the immunoactivating Fc domain-binding molecule.

71. An immunoactivating fragment crystallizable (Fc) domain-binding molecule produced by the method of claim 70.

72. A pharmaceutical composition comprising an immunoactivating Fc domain binding molecule according to any one of claims 1 to 66 and a pharmaceutically acceptable carrier.

73. An immune-activating Fc domain binding molecule according to any one of claims 1 to 66, or a pharmaceutical composition according to claim 72, for use as a pharmaceutical.

74. An immune-activating Fc domain binding molecule according to any one of claims 1 to 66 or a pharmaceutical composition according to claim 72, for use in the treatment of a disease in an individual requiring treatment of the disease.

75. The immunoactivating Fc domain conjugating molecule or pharmaceutical composition for use according to claim 74, wherein the disease is cancer.

76. An immunoactivating Fc domain binding molecule according to any one of claims 1 to 66, or a pharmaceutical composition according to claim 72, for use in the treatment of a disease in an individual requiring treatment of the disease, used in combination with a targeted antibody containing the target Fc domain.

77. The immunoactivating Fc domain binding molecule or pharmaceutical composition for use according to claim 76, wherein the disease is cancer.

78. The immunoactivating Fc domain binding molecule according to claim 76 or 77, wherein the targeted antibody is capable of specific binding to a target antigen.

79. Use of an immunoactivating Fc domain binding molecule according to any one of claims 1 to 66 for manufacturing a pharmaceutical for the treatment of a disease in an individual requiring treatment of the disease.

80. A pharmaceutical for treating a disease in an individual, comprising a composition containing an immune-activating Fc domain binding molecule according to any one of claims 1 to 66 in a pharmaceutically acceptable form, A pharmaceutical product comprising administering a therapeutically effective amount of the composition to the individual.

81. The use according to claim 79 or the pharmaceutical product according to claim 80, wherein the disease is cancer.

82. (a) A composition comprising, in a pharmaceutically acceptable form, an immunoactivating Fc domain binding molecule according to any one of claims 1 to 66; (b) A composition comprising a targeted antibody containing the target Fc domain; or, (c) the above (a) and (b) A pharmaceutical product for treating diseases in an individual, including, The composition containing an immune-activating Fc domain binding molecule in a therapeutically effective amount is administered to the individual, and The composition comprising a therapeutically effective amount of the targeted antibody containing the target Fc domain is administered to the individual. Pharmaceuticals.

83. The pharmaceutical product according to claim 81, wherein the disease is cancer.

84. The pharmaceutical product according to claim 82 or 83, wherein the targeted antibody is capable of specific binding to a target antigen.

85. The pharmaceutical product according to any one of claims 82 to 84, wherein the immune-activating Fc domain binding molecule is administered before, after, or simultaneously with the antibody containing the target Fc domain.

86. (a) The immunoactivating Fc domain binding molecule according to any one of claims 1 to 66; (b) A composition comprising a targeted antibody containing the target Fc domain; or, (c) the above (a) and (b) A pharmaceutical agent for inducing cell lysis, including The aforementioned cells are contacted with an immunoactivating Fc domain binding molecule according to any one of claims 1 to 66 and a targeted antibody containing the target Fc domain in the presence of T cells, and the targeted antibody is capable of specific binding to an antigen on the cells, wherein the cells are a pharmaceutical product.

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