Impact of tissue penetration and albumin binding on design of T cell targeted bispecific agents

Abstract

Bispecific agents are a rapidly growing class of cancer therapeutics, and immune targeted bispecific agents have the potential to expand functionality well beyond monoclonal antibody agents. Humabodies⁎ are fully human single domain antibodies that can be linked in a modular fashion to form multispecific therapeutics. However, the effect of heterogeneous delivery on the efficacy of crosslinking bispecific agents is currently unclear. In this work, we utilize a PSMA-CD137 Humabody with an albumin binding half-life extension (HLE) domain to determine the impact of tissue penetration on T cell activating bispecific agents. Using heterotypic spheroids, we demonstrate that increased tissue penetration results in higher T cell activation at sub-saturating concentrations. Next, we tested the effect of two different albumin binding moieties on tissue distribution using albumin-specific HLE domains with varying affinities for albumin and a non-specific lipophilic dye. The results show that a specific binding mechanism to albumin does not influence tissue penetration, but a non-specific mechanism reduced both spheroid uptake and distribution in the presence of albumin. These results highlight the potential importance of tissue penetration on bispecific agent efficacy and describe how the design parameters including albumin-binding domains can be selected to maximize the efficacy of bispecific agents.

Keywords: Bispecific antibodies; Heterotypic spheroids; Single domain antibodies; T-cell activating agents; Tumoral distribution.

Fig. 1

Binding Affinity and Mechanism. Measured binding affinity of PSMA and CD137 domains on cells (A). Synapse binding between T cells and cancer cells clusters the receptor to drive signaling (B). It is assumed that albumin binding does not interfere with clustering for these agents.

Fig. 2

Heterotypic spheroids for T cell activation. (A) Heterogeneous Humabody distribution may leave T cells in the center inactivated (left) relative to more homogeneous distribution (middle). However, migrating T cells and/or avidity increasing binding in the synapse may compensate for poor distribution (right). (B) T cell activation from 2D and 3D experiments demonstrated higher concentrations are needed for full activation in 3D culture, indicating tissue penetration is having an impact. The 3D spheroid saturation point is marked with a dotted line. (C) Images of heterotypic 22Rv1 spheroids show cell nuclei (blue), CD45 to mark T cells (magenta) or Humabody (green). An example of concentrated Humabody around a T cell marked with an arrow versus a T cell without concentrated Humabody marked, arrowhead. (D) Tissue gradients are steeper in the high expression DU145 spheroids, but T cells were mainly incorporated along the periphery, preventing measurement of activation in the middle of the spheroid. * p < 0.05.

Fig. 3

Tissue penetration of Humabodies with and without albumin in spheroids. Spheroids show cell nuclei (blue) and fluorescent drug (green). Incubations included 20 nM J591 antibody, monovalent-HLE Humabody, PSMA-CD137 with MSA binding HLE (mHLE) incubated with and without MSA, PSMA-CD137 with HSA binding HLE (hHLE) incubated with and without HSA (A). Euclidean distance mapping shows the fluorescent intensity versus distance from the spheroid edge in μm (B-D), indicating that specific albumin binding is not impacting tissue penetration to the spheroid center. The MSA binders (Fig. 2B) had lower maximum signal with the presence of MSA (p=0.002), but HSA binders did not have differences in the maximum signal (p = 0.85). *, p < 0.05.

Fig. 4

Impact of non-specific albumin binding on tissue penetration in spheroids. Spheroids were incubated for 24 h (A) or 6 h (B) with 20 nM Humabody labeled with lipophilic dye, Cy5.5. Euclidean distance mapping shows fluorescence intensity for each condition as a function of distance from spheroid edge. The non-specific interaction with albumin reduces the total uptake and penetration depth within the spheroids. **, p < 0.000001.

Fig. 5

Simulations of reversible Humabody binding via specific or non-specific mechanisms. Non-specific mechanisms can interact with multiple albumin domains and other proteins/membrans versus a single epitope (A). Schematic that displays how Humabody-albumin complexes may diffuse more slowly through tissue and potentially limit tissue penetration compared to free Humabody (B). A schematic of the spheroid simulations, which include diffusion, binding to the cell surface, binding to albumin, internalization, and degradation (C). Simulation results displaying concentration of Humabody versus distance from the spheroid edge with and without albumin binding (D).

Fig. 6

Confirmation of efficient tumor tissue penetration in vivo. Fluorescent tumor images show (A) J591 antibody or (B) Humabody (green) around functional vessels (blue) and CD31 stain (red) 24 h after a 0.7 nmol dose in nude mice (representative images from n=3 mice). Images were collected with different acquisition settings and displayed with different contrasts due to large differences in fluorescent intensity.