CD8+ T cell priming that is required for curative intratumorally anchored anti-4-1BB immunotherapy is constrained by Tregs.

Although co-stimulation of T cells with agonist antibodies targeting 4-1BB (CD137) improves antitumor immune responses in preclinical studies, clinical success has been limited by on-target, off-tumor activity. Here, we report the development of a tumor-anchored ɑ4-1BB agonist (ɑ4-1BB-LAIR), which consists of a ɑ4-1BB antibody fused to the collagen-binding protein LAIR. While combination treatment with an antitumor antibody (TA99) shows only modest efficacy, simultaneous depletion of CD4+ T cells boosts cure rates to over 90% of mice. Mechanistically, this synergy depends on ɑCD4 eliminating tumor draining lymph node regulatory T cells, resulting in priming and activation of CD8+ T cells which then infiltrate the tumor microenvironment. The cytotoxic program of these newly primed CD8+ T cells is then supported by the combined effect of TA99 and ɑ4-1BB-LAIR. The combination of TA99 and ɑ4-1BB-LAIR with a clinically approved ɑCTLA-4 antibody known for enhancing T cell priming results in equivalent cure rates, which validates the mechanistic principle, while the addition of ɑCTLA-4 also generates robust immunological memory against secondary tumor rechallenge. Thus, our study establishes the proof of principle for a clinically translatable cancer immunotherapy.

Both intratumoral regulatory T cell depletion and CTLA-4 antagonism are required for maximum efficacy of anti-CTLA-4 antibodies.

Anti-CTLA-4 antibodies have successfully elicited durable tumor regression in the clinic; however, long-term benefit is limited to a subset of patients for select cancer indications. The incomplete understanding of their mechanism of action has hindered efforts at improvement, with conflicting hypotheses proposing either antagonism of the CTLA-4:B7 axis or Fc effector-mediated regulatory T cell (Treg) depletion governing efficacy. Here, we report the engineering of a nonantagonistic CTLA-4 binding domain (b1s1e2) that depletes intratumoral Tregs as an Fc fusion. Comparison of b1s1e2-Fc to 9d9, an antagonistic anti-CTLA-4 antibody, allowed for interrogation of the separate contributions of CTLA-4 antagonism and Treg depletion to efficacy. Despite equivalent levels of intratumoral Treg depletion, 9d9 achieved more long-term cures than b1s1e2-Fc in MC38 tumors, demonstrating that CTLA-4 antagonism provided additional survival benefit. Consistent with prior reports that CTLA-4 antagonism enhances priming, treatment with 9d9, but not b1s1e2-Fc, increased the percentage of activated T cells in the tumor-draining lymph node (tdLN). Treg depletion with either construct was restricted to the tumor due to insufficient surface CTLA-4 expression on Tregs in other compartments. Through intratumoral administration of diphtheria toxin in Foxp3-DTR mice, we show that depletion of both intratumoral and nodal Tregs provided even greater survival benefit than 9d9, consistent with Treg-driven restraint of priming in the tdLN. Our data demonstrate that anti-CTLA-4 therapies require both CTLA-4 antagonism and intratumoral Treg depletion for maximum efficacy-but that potential future therapies also capable of depleting nodal Tregs could show efficacy in the absence of CTLA-4 antagonism.

Tregs constrain CD8+ T cell priming required for curative intratumorally anchored anti-4-1BB immunotherapy.

Although co-stimulation of T cells with agonist antibodies targeting 4-1BB (CD137) improves antitumor immune responses in preclinical studies, clinical development has been hampered by on-target, off-tumor toxicity. Here, we report the development of a tumor-anchored ɑ4-1BB agonist (ɑ4-1BB-LAIR), which consists of an ɑ4-1BB antibody fused to the collagen binding protein LAIR. While combination treatment with an antitumor antibody (TA99) displayed only modest efficacy, simultaneous depletion of CD4+ T cells boosted cure rates to over 90% of mice. We elucidated two mechanisms of action for this synergy: ɑCD4 eliminated tumor draining lymph node Tregs, enhancing priming and activation of CD8+ T cells, and TA99 + ɑ4-1BB-LAIR supported the cytotoxic program of these newly primed CD8+ T cells within the tumor microenvironment. Replacement of ɑCD4 with ɑCTLA-4, a clinically approved antibody that enhances T cell priming, produced equivalent cure rates while additionally generating robust immunological memory against secondary tumor rechallenge.

Alum-anchored intratumoral retention improves the tolerability and antitumor efficacy of type I interferon therapies.

Effective antitumor immunity in mice requires activation of the type I interferon (IFN) response pathway. IFNα and IFNß therapies have proven promising in humans, but suffer from limited efficacy and high toxicity. Intratumoral IFN retention ameliorates systemic toxicity, but given the complexity of IFN signaling, it was unclear whether long-term intratumoral retention of type I IFNs would promote or inhibit antitumor responses. To this end, we compared the efficacy of IFNα and IFNß that exhibit either brief or sustained retention after intratumoral injection in syngeneic mouse tumor models. Significant enhancement in tumor retention, mediated by anchoring these IFNs to coinjected aluminum-hydroxide (alum) particles, greatly improved both their tolerability and efficacy. The improved efficacy of alum-anchored IFNs could be attributed to sustained pleiotropic effects on tumor cells, immune cells, and nonhematopoietic cells. Alum-anchored IFNs achieved high cure rates of B16F10 tumors upon combination with either anti-PD-1 antibody or interleukin-2. Interestingly however, these alternative combination immunotherapies yielded disparate T cell phenotypes and differential resistance to tumor rechallenge, highlighting important distinctions in adaptive memory formation for combinations of type I IFNs with other immunotherapies.

An affinity threshold for maximum efficacy in anti-PD-1 immunotherapy.

Monoclonal antibodies targeting the programmed cell death protein 1 (PD-1) remain the most prevalent cancer immunotherapy both as a monotherapy and in combination with additional therapies. Despite the extensive success of anti-PD-1 monoclonal antibodies in the clinic, the experimental relationship between binding affinity and functional potency for anti-PD-1 antibodies in vivo has not been reported. Anti-PD-1 antibodies with higher and lower affinity than nivolumab or pembrolizumab are entering the clinic and show varied preclinical efficacy. Here, we explore the role of broad-ranging affinity variation within a single lineage in a syngeneic immunocompetent mouse model. By developing a panel of murine anti-PD-1 antibodies with varying affinity (ranging from KD = 20 pM - 15 nM), we find that there is a threshold affinity required for maximum efficacy at a given dose in the treatment of the MC38 adenocarcinoma model with anti-PD-1 immunotherapy. Physiologically based pharmacokinetic modeling complements interpretation of the experimental results and highlights the direct relationship between dose, affinity, and PD-1 target saturation in the tumor.

Yeast Surface Display for Protein Engineering: Library Generation, Screening, and Affinity Maturation.

Yeast surface display is a powerful directed evolution method for developing and engineering protein molecules to attain desired properties. Here, updated protocols are presented for purposes of identification of lead binders and their affinity maturation. Large libraries are screened by magnetic bead selections followed by flow cytometric selections. Upon identification and characterization of single clones, their affinities are improved by an iterative process of mutagenesis and fluorescence-activated cell sorting.