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Cited by 12Cancer Immunotherapy and BiomarkersMonoclonal and Polyclonal Antibodies ResearchImmunotherapy and Immune Responses

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NI-3201 Is a Bispecific Antibody Mediating PD-L1–Dependent CD28 Co-stimulation on T Cells for Enhanced Tumor Control

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Majocchi, Sara, Lloveras, Pauline, Nouveau, Lise, Legrand, Margaux, Viandier, Alizee, Malinge, Pauline, Charreton, Maud, Raymond, Cecile, Pace, Emily A., Millard, Bjorn L., Svensson, L. Anders, Kelpšas, Vinardas, Anceriz, Nadia, Salgado-Pires, Susana, Daubeuf, Bruno, Magistrelli, Giovanni, Gueneau, Franck, Moine, Valéry, Masternak, Krzysztof, Shang, Limin, Fischer, Nicolas, Ferlin, Walter G.·2025·Cancer Immunology Research
DOI 10.1158/2326-6066.cir-24-0298PMC PMC11876958

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Abstract

Despite advances in cancer immunotherapy, such as targeting the PD-1/PD-L1 axis, a substantial number of patients harbor tumors that are resistant or relapse. Selective engagement of T-cell co-stimulatory molecules with bispecific antibodies may offer novel therapeutic options by enhancing signal 1–driven activation occurring via T-cell receptor engagement. In this study, we report the development and preclinical characterization of NI-3201, a PD-L1×CD28 bispecific antibody generated on the κλ-body platform that was designed to promote T-cell activity and antitumor function through a dual mechanism of action. We confirmed that NI-3201 blocks the PD-L1/PD-1 immune checkpoint pathway and conditionally provides T-cell co-stimulation via CD28 (signal 2) when engaging PD-L1+ tumors or immune cells. In systems with signal 1–primed T cells, NI-3201 enhanced potent effector functionality: in vitro through antigen-specific recall assays with cytomegalovirus-specific T cells and in vivo by inducing tumor regression and immunologic memory in tumor-associated antigen–expressing MC38 syngeneic mouse models. When T-cell engagers were used to provide synthetic signal 1, the combination with NI-3201 resulted in synergistic T cell–dependent cytotoxicity and potent antitumor activity in two humanized mouse tumor models. Nonhuman primate safety assessments showed favorable tolerability and pharmacokinetics at pharmacologically active doses. Quantitative systems pharmacology modeling predicted that NI-3201 exposure results in antitumor activity in patients, but this remains to be investigated. Overall, this study suggests that by combining PD-L1 blockade with safe and effective CD28 co-stimulation, NI-3201 has the potential to improve cancer immunotherapy outcomes, and the clinical development of NI-3201 for PD-L1+ solid tumors is planned.

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Figure 1. The anti-CD28 AI3 arm is not an SA and binds to a distal epitope on CD28, potentially competing with endogenous ligands. A, Binding of the anti-CD28 antibody AI3 either in a bivalent or monovalent format to CD28+ Jurkat (left) or CD28− J.RT3-T3.5 (right) cells, as determined by flow cytometry (n = 3). B, Proliferation of CD4+ T cells following incubation of PBMCs derived from healthy donors on culture plates coated with the mentioned antibodies using wet coating procedures. Proliferation was quantified by dye dilution using flow cytometry. Irrelevant human IgG1 was used as the negative control, whereas the anti-CD3 antibody OKT3 and TGN1412 served as positive controls. Each color represents the average proliferation based on technical triplicates obtained with a series of different PBMC donors (n = 20), with the median for each group shown as a bar. C, Epitope of the AI3 arm on human CD28, as determined by crystallography studies based on distance calculations between AI3 and CD28 residues. Residues of the AI3 epitope that are unique to CD28 are shown in purple, whereas residues that are shared between CD28 and CTLA-4 are shown in aqua blue. The CD28 ligand–binding region corresponding to the MYPPPY motif is highlighted by a red dashed line. D, Crystal structure of the AI3 Fab complexed to the extracellular domain of human CD28 shows the interactions between CD28 (pink, with the MYPPPY motif highlighted by a cloud) and the AI3 complementarity-determining regions (CDRH1–3 in green, cyan, and blue, and CDRL1–3 in red, orange, and yellow, respectively). The antibody framework regions are shown in gray. E, Concentration-dependent decrease in luminescence in a CD28 blockade reporter assay aiming at quantifying the capacity of the AI3 arm to prevent the binding of CD80 and CD86 to CD28. AI3 was tested as a bivalent antibody (AI3 bivalent) and monovalent bsAb (two different constructs termed AI3 monovalent #1 and #2). A blocking anti-CD28 mAb and an irrelevant mAb were used as positive and negative controls, respectively. Percentage of reporter gene expression reported relative to the maximal luminescence obtained in the absence of Abs. The mean values of technical duplicates are shown as mean ± SD (n = 3). Max, maximal; MFI, mean fluorescence intensity; ns, not significant.
Figure 1. The anti-CD28 AI3 arm is not an SA and binds to a distal epitope on CD28, potentially competing with endogenous ligands. A, Binding of the anti-CD28 antibody AI3 either in a bivalent or monovalent format to CD28+ Jurkat (left) or CD28− J.RT3-T3.5 (right) cells, as determined by flow cytometry (n = 3). B, Proliferation of CD4+ T cells following incubation of PBMCs derived from healthy donors on culture plates coated with the mentioned antibodies using wet coating procedures. Proliferation was quantified by dye dilution using flow cytometry. Irrelevant human IgG1 was used as the negative control, whereas the anti-CD3 antibody OKT3 and TGN1412 served as positive controls. Each color represents the average proliferation based on technical triplicates obtained with a series of different PBMC donors (n = 20), with the median for each group shown as a bar. C, Epitope of the AI3 arm on human CD28, as determined by crystallography studies based on distance calculations between AI3 and CD28 residues. Residues of the AI3 epitope that are unique to CD28 are shown in purple, whereas residues that are shared between CD28 and CTLA-4 are shown in aqua blue. The CD28 ligand–binding region corresponding to the MYPPPY motif is highlighted by a red dashed line. D, Crystal structure of the AI3 Fab complexed to the extracellular domain of human CD28 shows the interactions between CD28 (pink, with the MYPPPY motif highlighted by a cloud) and the AI3 complementarity-determining regions (CDRH1–3 in green, cyan, and blue, and CDRL1–3 in red, orange, and yellow, respectively). The antibody framework regions are shown in gray. E, Concentration-dependent decrease in luminescence in a CD28 blockade reporter assay aiming at quantifying the capacity of the AI3 arm to prevent the binding of CD80 and CD86 to CD28. AI3 was tested as a bivalent antibody (AI3 bivalent) and monovalent bsAb (two different constructs termed AI3 monovalent #1 and #2). A blocking anti-CD28 mAb and an irrelevant mAb were used as positive and negative controls, respectively. Percentage of reporter gene expression reported relative to the maximal luminescence obtained in the absence of Abs. The mean values of technical duplicates are shown as mean ± SD (n = 3). Max, maximal; MFI, mean fluorescence intensity; ns, not significant.CC BY-NC-ND · PubMed Central
Figure 2. NI-3201 mediates CD28 co-stimulation upon PD-L1 blockade. A, NI-3201 is a κλ body composed of the anti-CD28 κ-arm AI3 and the anti–PD-L1 λ-arm S79. The κ and λ light chains drive the specificity of each arm, whereas the two identical HCs were engineered to prevent binding to FcγRs by inserting the triple LALAPA mutation into the human IgG1 Fc. B and C, NI-3201–intended MoA include the blockade of the PD-1/PD-L1 interaction with subsequent CD28-mediated co-stimulation of T cells, both on PD-L1+ tumor cells in the TME (B) and on PD-L1+ immune cells [i.e., dendritic cell (DC)] in lymph nodes (C). D, Binding of increasing concentrations of NI-3201 to PD-L1+ IFNγ-primed HPAC cells as assessed by flow cytometry. A monovalent S79 bsAb and bivalent S79 mAb were used as controls (n = 3). E, IL2 secretion induced by either TGN1412 or NI-3201 when incubated with HD precultured PBMC according to the “RESTORE” protocol (26). The three donors (D) highlighted variations in the amplitude of the response induced by TGN1412. The mean values of technical duplicates are shown as mean ± SD for three representative donors (of the nine donors tested). F, NFAT-dependent luciferase activity in a reporter assay designed to respond to PD-L1 blockade. Fold increase was reported after 6 hours of stimulation relative to baseline luminescence in the absence of anti–PD-L1 antibodies with increasing concentrations of NI-3201, monovalent and bivalent controls (for both AI3 and S79 arms), and atezolizumab or pembrolizumab (both serving as positive controls). The mean values of technical duplicates are shown as mean ± SD (n = 3). MFI, mean fluorescence intensity. (A, B, and C created with BioRender.com.)
Figure 2. NI-3201 mediates CD28 co-stimulation upon PD-L1 blockade. A, NI-3201 is a κλ body composed of the anti-CD28 κ-arm AI3 and the anti–PD-L1 λ-arm S79. The κ and λ light chains drive the specificity of each arm, whereas the two identical HCs were engineered to prevent binding to FcγRs by inserting the triple LALAPA mutation into the human IgG1 Fc. B and C, NI-3201–intended MoA include the blockade of the PD-1/PD-L1 interaction with subsequent CD28-mediated co-stimulation of T cells, both on PD-L1+ tumor cells in the TME (B) and on PD-L1+ immune cells [i.e., dendritic cell (DC)] in lymph nodes (C). D, Binding of increasing concentrations of NI-3201 to PD-L1+ IFNγ-primed HPAC cells as assessed by flow cytometry. A monovalent S79 bsAb and bivalent S79 mAb were used as controls (n = 3). E, IL2 secretion induced by either TGN1412 or NI-3201 when incubated with HD precultured PBMC according to the “RESTORE” protocol (26). The three donors (D) highlighted variations in the amplitude of the response induced by TGN1412. The mean values of technical duplicates are shown as mean ± SD for three representative donors (of the nine donors tested). F, NFAT-dependent luciferase activity in a reporter assay designed to respond to PD-L1 blockade. Fold increase was reported after 6 hours of stimulation relative to baseline luminescence in the absence of anti–PD-L1 antibodies with increasing concentrations of NI-3201, monovalent and bivalent controls (for both AI3 and S79 arms), and atezolizumab or pembrolizumab (both serving as positive controls). The mean values of technical duplicates are shown as mean ± SD (n = 3). MFI, mean fluorescence intensity. (A, B, and C created with BioRender.com.)CC BY-NC-ND · PubMed Central
Figure 3. NI-3201 provides signal 2 and activates T cells in an antigen-dependent manner in an adapted mixed lymphocyte reaction (MLR) assay and a CMV recall assay. A, Amplification of a mixed lymphocyte reaction mediated by NI-3201, as determined by the increase in IL2 secretion by CD4+ T cells (responders) cultured with MoDCs (stimulators, from a different donor) in the presence of staphylococcal enterotoxin A (SEA). The cells were co-cultured for 4 days. Atezolizumab was used as the benchmark; the S79 monovalent and bivalent mAbs served as comparators, whereas the AI3 monovalent and an irrelevant IgG were used as negative controls. Pembrolizumab was also tested to assess the effects of PD-1 blockade, alone or in combination with the mentioned antibodies. The mean values of technical duplicates are shown as mean ± SD (n = 4 different responder/stimulator pairs). B, Expansion of NLV-specific CD8 memory T cells after incubation of PBMCs from CMV+ donors with IFNγ-primed MDA-MB-231 cells ectopically loaded with a pool of CMV peptides in the presence of increasing concentrations of NI-3201. E:T ratio = 10:1. The two donors highlighted variations in the amplitude of the response induced by NI-3201. As control, the expansion induced by NI-3201 in the presence of non–CMV-loaded target cells or induced by an isotype control in the presence of CMV peptide–loaded target cells is shown. Expansion was quantified using a specific NLV tetramer (n = 2 donors tested; 9 additional CMV+ donors tested using NLV-loaded target cells). DR, dose-response.
Figure 3. NI-3201 provides signal 2 and activates T cells in an antigen-dependent manner in an adapted mixed lymphocyte reaction (MLR) assay and a CMV recall assay. A, Amplification of a mixed lymphocyte reaction mediated by NI-3201, as determined by the increase in IL2 secretion by CD4+ T cells (responders) cultured with MoDCs (stimulators, from a different donor) in the presence of staphylococcal enterotoxin A (SEA). The cells were co-cultured for 4 days. Atezolizumab was used as the benchmark; the S79 monovalent and bivalent mAbs served as comparators, whereas the AI3 monovalent and an irrelevant IgG were used as negative controls. Pembrolizumab was also tested to assess the effects of PD-1 blockade, alone or in combination with the mentioned antibodies. The mean values of technical duplicates are shown as mean ± SD (n = 4 different responder/stimulator pairs). B, Expansion of NLV-specific CD8 memory T cells after incubation of PBMCs from CMV+ donors with IFNγ-primed MDA-MB-231 cells ectopically loaded with a pool of CMV peptides in the presence of increasing concentrations of NI-3201. E:T ratio = 10:1. The two donors highlighted variations in the amplitude of the response induced by NI-3201. As control, the expansion induced by NI-3201 in the presence of non–CMV-loaded target cells or induced by an isotype control in the presence of CMV peptide–loaded target cells is shown. Expansion was quantified using a specific NLV tetramer (n = 2 donors tested; 9 additional CMV+ donors tested using NLV-loaded target cells). DR, dose-response.CC BY-NC-ND · PubMed Central
Figure 4. NI-3201 shows strong single-agent antitumor activity and induction of immunological memory in immunocompetent huCD28 mice. A, Human (hu)PD-L1-MC38 cells were subcutaneously engrafted into the rear right flank of huCD28-Tg mice. When the average tumor volume was close to 100 mm3, mice were randomized into 3 groups of 10 animals and treated weekly for a total of 3 injections of vehicle (PBS), atezolizumab (5 mg/kg, bivalent anti–PD-L1 antibody), or NI-3201 (10 mg/kg, monovalent engagement of each target). The experiment was terminated on day 28 after engraftment. B, Average tumor volume in different treatment groups ± SEM. C, Probability of survival of mice enrolled in the different study groups. Mice in the atezolizumab group, which unexpectedly died, were excluded from this analysis. Statistical analysis was performed using the log-rank (Mantel–Cox) test with Bonferroni correction. P value (NI-3201 vs. atezolizumab) = 0.0035; P value (NI-3201 vs. vehicle) = 0.0012. D, Mouse cytokine secretion 6 hours after first injection, with IFNγ, IL2, IL6, and TNFα, respectively. Statistical analysis was performed using one-way ANOVA coupled with the Tukey multiple comparison test. Mean ± SD values are shown. E, Mice from the NI-3201 treatment group that survived the primary tumor challenge with huPD-L1-MC38 (n = 8/10) were rechallenged with WT-MC38 cells on the opposite flank. Naïve mice (n = 8) served as tumor growth controls. The average tumor volume of rechallenged mice vs. tumor-naïve mice (± SEM) is shown. F, huCD28-Tg mice were engrafted subcutaneously with MC38 cells expressing human CEA (huCEA-MC38) and treated three times during 7 days with NI-3201 starting either on day 1 (early treatment settings, n = 13) or when the tumor volume reached 50 mm3 (n = 13). As control, mice were treated with vehicle (PBS, n = 14). G, Survival probability of mice receiving NI-3201 treatments compared with vehicle control. Statistical analysis by the log-rank (Mantel–Cox) test with Bonferroni correction. P value (vehicle vs. NI-3201 early) = 0.0008; P value (vehicle vs. NI-3201 late) < 0.0001; P value (NI-3201 early vs. NI-3201 late) = 0.0672. H, Tumor growth of individual mice that rejected the primary tumor challenge when engrafted on the opposite flank with WT-MC38. Naïve mice served as the tumor growth control. I, huCD28-Tg mice were engrafted subcutaneously with OVA-MC38 and treated twice on days 7 and 11 after engraftment before being euthanized for TME and dLN analyses on day 15. J, Average tumor volume ± SEM. t test performed on tumor volume on day 14. K, Immunophenotyping of tumor-infiltrating lymphocytes, with the number of CD3+, CD4+, or CD8+ T cells per milligram of tumor (top row) and the relative frequency of central memory T cells (TCM, gated as CD44+CD62L+) and effector T cells (TEff, gated as CD44+CD62L−) in the CD4+ and CD8+ subsets (bottom row). Mean ± SD values are shown. L, huCD28-Tg mice were engrafted subcutaneously with OVA-MC38 cells and treated twice on days 7 and 11 after engraftment ± administration of FTY720 to impair lymphocyte trafficking. M, Survival probability of mice receiving NI-3201 compared with vehicle control, with or without concomitant FTY720 administration. Statistical analysis by the log-rank (Mantel–Cox) test with Bonferroni correction. P value (NI-3201 vs. vehicle) = 0.0086; P value (NI-3201 vs. FTY720) = 0.0082; P value (NI-3201 vs. NI-3201 + FTY720) = 0.0425; P value (FTY720 vs. NI-3201 + FTY720) = 0.0328; P value (NI-3201 + FTY720 vs. vehicle) = 0.0975; P value (vehicle vs. FTY720) = 0.7232. *, P < 0.05; **, P < 0.01; ***, P < 0.001. ns, not significant.
Figure 4. NI-3201 shows strong single-agent antitumor activity and induction of immunological memory in immunocompetent huCD28 mice. A, Human (hu)PD-L1-MC38 cells were subcutaneously engrafted into the rear right flank of huCD28-Tg mice. When the average tumor volume was close to 100 mm3, mice were randomized into 3 groups of 10 animals and treated weekly for a total of 3 injections of vehicle (PBS), atezolizumab (5 mg/kg, bivalent anti–PD-L1 antibody), or NI-3201 (10 mg/kg, monovalent engagement of each target). The experiment was terminated on day 28 after engraftment. B, Average tumor volume in different treatment groups ± SEM. C, Probability of survival of mice enrolled in the different study groups. Mice in the atezolizumab group, which unexpectedly died, were excluded from this analysis. Statistical analysis was performed using the log-rank (Mantel–Cox) test with Bonferroni correction. P value (NI-3201 vs. atezolizumab) = 0.0035; P value (NI-3201 vs. vehicle) = 0.0012. D, Mouse cytokine secretion 6 hours after first injection, with IFNγ, IL2, IL6, and TNFα, respectively. Statistical analysis was performed using one-way ANOVA coupled with the Tukey multiple comparison test. Mean ± SD values are shown. E, Mice from the NI-3201 treatment group that survived the primary tumor challenge with huPD-L1-MC38 (n = 8/10) were rechallenged with WT-MC38 cells on the opposite flank. Naïve mice (n = 8) served as tumor growth controls. The average tumor volume of rechallenged mice vs. tumor-naïve mice (± SEM) is shown. F, huCD28-Tg mice were engrafted subcutaneously with MC38 cells expressing human CEA (huCEA-MC38) and treated three times during 7 days with NI-3201 starting either on day 1 (early treatment settings, n = 13) or when the tumor volume reached 50 mm3 (n = 13). As control, mice were treated with vehicle (PBS, n = 14). G, Survival probability of mice receiving NI-3201 treatments compared with vehicle control. Statistical analysis by the log-rank (Mantel–Cox) test with Bonferroni correction. P value (vehicle vs. NI-3201 early) = 0.0008; P value (vehicle vs. NI-3201 late) < 0.0001; P value (NI-3201 early vs. NI-3201 late) = 0.0672. H, Tumor growth of individual mice that rejected the primary tumor challenge when engrafted on the opposite flank with WT-MC38. Naïve mice served as the tumor growth control. I, huCD28-Tg mice were engrafted subcutaneously with OVA-MC38 and treated twice on days 7 and 11 after engraftment before being euthanized for TME and dLN analyses on day 15. J, Average tumor volume ± SEM. t test performed on tumor volume on day 14. K, Immunophenotyping of tumor-infiltrating lymphocytes, with the number of CD3+, CD4+, or CD8+ T cells per milligram of tumor (top row) and the relative frequency of central memory T cells (TCM, gated as CD44+CD62L+) and effector T cells (TEff, gated as CD44+CD62L−) in the CD4+ and CD8+ subsets (bottom row). Mean ± SD values are shown. L, huCD28-Tg mice were engrafted subcutaneously with OVA-MC38 cells and treated twice on days 7 and 11 after engraftment ± administration of FTY720 to impair lymphocyte trafficking. M, Survival probability of mice receiving NI-3201 compared with vehicle control, with or without concomitant FTY720 administration. Statistical analysis by the log-rank (Mantel–Cox) test with Bonferroni correction. P value (NI-3201 vs. vehicle) = 0.0086; P value (NI-3201 vs. FTY720) = 0.0082; P value (NI-3201 vs. NI-3201 + FTY720) = 0.0425; P value (FTY720 vs. NI-3201 + FTY720) = 0.0328; P value (NI-3201 + FTY720 vs. vehicle) = 0.0975; P value (vehicle vs. FTY720) = 0.7232. *, P < 0.05; **, P < 0.01; ***, P < 0.001. ns, not significant.CC BY-NC-ND · PubMed Central
Figure 5. NI-3201 synergizes with a TCE to kill tumor cells in vitro and in vivo. A, TDCC of HPAC cells induced by NI-3201 when combined with a CEA×CD3 TCE. PBMCs were added to target cells at different final E:T ratios (10:1, 3:1, 1:1, and 1:3) and co-cultured in the presence of a dose range of TCE and a fixed dose of NI-3201 (2.5 μg/mL). Single-agent NI-3201 was used as the negative control. Target cell death was assessed after 6 days of incubation. The mean values of technical duplicates are shown as mean ± SD (n = 2). t test performed on the AUC values from TCE alone or in combination with NI-3201. B, Average volume (± SEM) of HPAF-II tumors implanted subcutaneously in the flank of BRGSF-HIS mice. Treatment was administered on days 8 (average tumor volume: 65 mm3), 11, and 14 and consisted of TCE (10 mg/kg) and/or NI-3201 (10 mg/kg) or vehicle (PBS; n = 10 each). Statistical analysis was performed using one-way ANOVA coupled with the Tukey multiple comparison test on AUC7–23days. P value (vehicle vs. TCE + NI-3201) = 0.0229; P value (TCE vs. TCE + NI-3201) = 0.0479. C, Tumor growth of individual mice belonging to the vehicle (left), TCE (center), or TCE + NI-3201 (right) groups. Animals marked with red dots were euthanized because of necrotic tumors. *, P < 0.05; **, P < 0.01.
Figure 5. NI-3201 synergizes with a TCE to kill tumor cells in vitro and in vivo. A, TDCC of HPAC cells induced by NI-3201 when combined with a CEA×CD3 TCE. PBMCs were added to target cells at different final E:T ratios (10:1, 3:1, 1:1, and 1:3) and co-cultured in the presence of a dose range of TCE and a fixed dose of NI-3201 (2.5 μg/mL). Single-agent NI-3201 was used as the negative control. Target cell death was assessed after 6 days of incubation. The mean values of technical duplicates are shown as mean ± SD (n = 2). t test performed on the AUC values from TCE alone or in combination with NI-3201. B, Average volume (± SEM) of HPAF-II tumors implanted subcutaneously in the flank of BRGSF-HIS mice. Treatment was administered on days 8 (average tumor volume: 65 mm3), 11, and 14 and consisted of TCE (10 mg/kg) and/or NI-3201 (10 mg/kg) or vehicle (PBS; n = 10 each). Statistical analysis was performed using one-way ANOVA coupled with the Tukey multiple comparison test on AUC7–23days. P value (vehicle vs. TCE + NI-3201) = 0.0229; P value (TCE vs. TCE + NI-3201) = 0.0479. C, Tumor growth of individual mice belonging to the vehicle (left), TCE (center), or TCE + NI-3201 (right) groups. Animals marked with red dots were euthanized because of necrotic tumors. *, P < 0.05; **, P < 0.01.CC BY-NC-ND · PubMed Central
Figure 6. NI-3201 displays favorable PK in cynomolgus monkeys. A, NI-3201 concentrations vs. time after intravenous administration at 0.57 (single) or 10 mg/kg (single or repeated) in cynomolgus monkeys (semilogarithmic scale). Asterisk (*) denotes animals receiving repeated 10-mg/kg injections that were sacrificed for histopathologic examination on day 15. The dotted line represents the lower limit of quantification (LLOQ) value (0.02 µg/mL). B–E, Animals from the 10-mg/kg treated group shown in (A) were bled several times for immunophenotyping. B, Frequency of CD3+ T cells among CD45+ leukocyte population. C, Frequency of proliferative CD3+ T cells based on Ki67 staining. D and E, Frequency of activated CD3+ T cells based on CD25 and ICOS staining, respectively. Mean ± SEM values are shown. MFI, mean fluorescence intensity.
Figure 6. NI-3201 displays favorable PK in cynomolgus monkeys. A, NI-3201 concentrations vs. time after intravenous administration at 0.57 (single) or 10 mg/kg (single or repeated) in cynomolgus monkeys (semilogarithmic scale). Asterisk (*) denotes animals receiving repeated 10-mg/kg injections that were sacrificed for histopathologic examination on day 15. The dotted line represents the lower limit of quantification (LLOQ) value (0.02 µg/mL). B–E, Animals from the 10-mg/kg treated group shown in (A) were bled several times for immunophenotyping. B, Frequency of CD3+ T cells among CD45+ leukocyte population. C, Frequency of proliferative CD3+ T cells based on Ki67 staining. D and E, Frequency of activated CD3+ T cells based on CD25 and ICOS staining, respectively. Mean ± SEM values are shown. MFI, mean fluorescence intensity.CC BY-NC-ND · PubMed Central
Figure 7. QSP modeling informs on favorable NI-3201 dosing in patients. A, Diagram of the human three-compartment, semimechanistic PK/PD model established for NI-3201 including intravenous dosing in the central compartment, drug-binding interactions, drug distribution between compartments, cell and protein homeostasis, and linear and nonlinear drug elimination. B, PK projection in the central (left), peripheral (center), and tumor (right) compartments in the three-compartment NI-3201 human model. C, Time series of the projected PD-L1 RO% in the tumor compartment for a single dose escalation of NI-3201 in the presence of different PD-L1 surface expression levels (10,000, left; 30,000, right). D, Time series of the projected trimer formation in the tumor compartment for a single dose escalation of NI-3201 in the presence of different PD-L1 surface expression levels (10,000, left; 30,000, right). kbirth, cell generation rate; kdeath, cell elimination rate; kendo, protein endocytosis rate; ksynth, protein synthesis rate; TMDD, target-mediated drug disposition.
Figure 7. QSP modeling informs on favorable NI-3201 dosing in patients. A, Diagram of the human three-compartment, semimechanistic PK/PD model established for NI-3201 including intravenous dosing in the central compartment, drug-binding interactions, drug distribution between compartments, cell and protein homeostasis, and linear and nonlinear drug elimination. B, PK projection in the central (left), peripheral (center), and tumor (right) compartments in the three-compartment NI-3201 human model. C, Time series of the projected PD-L1 RO% in the tumor compartment for a single dose escalation of NI-3201 in the presence of different PD-L1 surface expression levels (10,000, left; 30,000, right). D, Time series of the projected trimer formation in the tumor compartment for a single dose escalation of NI-3201 in the presence of different PD-L1 surface expression levels (10,000, left; 30,000, right). kbirth, cell generation rate; kdeath, cell elimination rate; kendo, protein endocytosis rate; ksynth, protein synthesis rate; TMDD, target-mediated drug disposition.CC BY-NC-ND · PubMed Central

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Metadata

Authors
Majocchi, S., Lloveras, P., Nouveau, L., Legrand, M., Viandier, A., Malinge, P., Charreton, M., Raymond, C., Pace, E. A., Millard, B. L., Svensson, L. A., Kelpšas, V., Anceriz, N., Salgado-Pires, S., Daubeuf, B., Magistrelli, G., Gueneau, F., Moine, V., Masternak, K., Shang, L., Fischer, N., & Ferlin, W. G.
Affiliations
  • Light Chain Bioscience (Switzerland) (CH)
  • 1Light Chain Bioscience – Novimmune SA, Geneva, Switzerland.
  • 2Residual Dynamics, Camano Island, Washington.
  • SARomics Biostructures (Sweden) (SE)
Corresponding author
Ferlin, Walter G.
ORCID
Venue
Cancer Immunology Research
Publisher
American Association for Cancer Research (AACR)
Published
2025-01-06
Volume
13
Issue
3
Pages
365-383
ISSN
  • 2326-6066 (ISSN-L)
  • 2326-6074
DOI
10.1158/2326-6066.cir-24-0298
PMC
PMC11876958
OpenAlex
W4406086774
Cited by
12
References
9
Citations by year
2026: 7 · 2025: 5
FWCI
4.94
Type
journal article
Language
en
Open access
Licence
Locations
  • Cancer Immunology Research (publishedVersion, cc-by-nc-nd)
  • PubMed Central (publishedVersion, cc-by-nc-nd)
  • PubMed (publishedVersion)
Funders
  • VINNOVA (2019‐02496, 2018-07152, 2018-04969)
  • Svenska Forskningsrådet Formas (2018-07152, 2019-02496)
  • Vetenskapsrådet (2018–04969, 07152, 2019–02496, 201807152, 2018-)
Topics
  • Health Sciences › Medicine › Oncology › Cancer Immunotherapy and Biomarkers
  • Life Sciences › Immunology and Microbiology › Immunology › Monoclonal and Polyclonal Antibodies Research
  • Life Sciences › Immunology and Microbiology › Immunology › Immunotherapy and Immune Responses
Keywords
bispecific antibodies, PD-L1, CD28, T cell costimulation, PD-1/PD-L1 blockade, T-cell engagers, T cell activation, antitumor immunity, MC38 colon carcinoma, pharmacokinetics, quantitative systems pharmacology, solid tumors
MeSH terms
Animals, Female, Humans, Immunotherapy, Lymphocyte Activation, Neoplasms, T-Lymphocytes, Antibodies, Bispecific, CD28 Antigens, Xenograft Model Antitumor Assays, Cell Line, Tumor, Mice, B7-H1 Antigen

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