Molecular cartography uncovers evolutionary and microenvironmental dynamics in sporadic colorectal tumors.

Colorectal cancer exhibits dynamic cellular and genetic heterogeneity during progression from precursor lesions toward malignancy. Analysis of spatial multi-omic data from 31 human colorectal specimens enabled phylogeographic mapping of tumor evolution that revealed individualized progression trajectories and accompanying microenvironmental and clonal alterations. Phylogeographic mapping ordered genetic events, classified tumors by their evolutionary dynamics, and placed clonal regions along global pseudotemporal progression trajectories encompassing the chromosomal instability (CIN+) and hypermutated (HM) pathways. Integrated single-cell and spatial transcriptomic data revealed recurring epithelial programs and infiltrating immune states along progression pseudotime. We discovered an immune exclusion signature (IEX), consisting of extracellular matrix regulators DDR1, TGFBI, PAK4, and DPEP1, that charts with CIN+ tumor progression, is associated with reduced cytotoxic cell infiltration, and shows prognostic value in independent cohorts. This spatial multi-omic atlas provides insights into colorectal tumor-microenvironment co-evolution, serving as a resource for stratification and targeted treatments.

A highly selective humanized DDR1 mAb reverses immune exclusion by disrupting collagen fiber alignment in breast cancer.

BACKGROUND: Immune exclusion (IE) where tumors deter the infiltration of immune cells into the tumor microenvironment has emerged as a key mechanism underlying immunotherapy resistance. We recently reported a novel role of discoidin domain-containing receptor 1 (DDR1) in promoting IE in breast cancer and validated its critical role in IE using neutralizing rabbit monoclonal antibodies (mAbs) in multiple mouse tumor models. METHODS: To develop a DDR1-targeting mAb as a potential cancer therapeutic, we humanized mAb9 with a complementarity-determining region grafting strategy. The humanized antibody named PRTH-101 is currently being tested in a Phase 1 clinical trial. We determined the binding epitope of PRTH-101 from the crystal structure of the complex between DDR1 extracellular domain (ECD) and the PRTH-101 Fab fragment with 3.15 Å resolution. We revealed the underlying mechanisms of action of PRTH-101 using both cell culture assays and in vivo study in a mouse tumor model. RESULTS: PRTH-101 has subnanomolar affinity to DDR1 and potent antitumor efficacy similar to the parental rabbit mAb after humanization. Structural information illustrated that PRTH-101 interacts with the discoidin (DS)-like domain, but not the collagen-binding DS domain of DDR1. Mechanistically, we showed that PRTH-101 inhibited DDR1 phosphorylation, decreased collagen-mediated cell attachment, and significantly blocked DDR1 shedding from the cell surface. Treatment of tumor-bearing mice with PRTH-101 in vivo disrupted collagen fiber alignment (a physical barrier) in the tumor extracellular matrix (ECM) and enhanced CD8+ T cell infiltration in tumors. CONCLUSIONS: This study not only paves a pathway for the development of PRTH-101 as a cancer therapeutic, but also sheds light on a new therapeutic strategy to modulate collagen alignment in the tumor ECM for enhancing antitumor immunity.

Nonclinical Development of SRK-181: An Anti-Latent TGFß1 Monoclonal Antibody for the Treatment of Locally Advanced or Metastatic Solid Tumors.

Checkpoint inhibitors offer a promising immunotherapy strategy for cancer treatment; however, due to primary or acquired resistance, many patients do not achieve lasting clinical responses. Recently, the transforming growth factor-ß (TGFß) signaling pathway has been identified as a potential target to overcome primary resistance, although the nonselective inhibition of multiple TGFß isoforms has led to dose-limiting cardiotoxicities. SRK-181 is a high-affinity, fully human antibody that selectively binds to latent TGFß1 and inhibits its activation. To support SRK-181 clinical development, we present here a comprehensive preclinical assessment of its pharmacology, pharmacokinetics, and safety across multiple species. In vitro studies showed that SRK-181 has no effect on human platelet function and does not induce cytokine release in human peripheral blood. Four-week toxicology studies with SRK-181 showed that weekly intravenous administration achieved sustained serum exposure and was well tolerated in rats and monkeys, with no treatment-related adverse findings. The no-observed-adverse-effect levels levels were 200 mg/kg in rats and 300 mg/kg in monkeys, the highest doses tested, and provide a nonclinical safety factor of up to 813-fold (based on Cmax) above the phase 1 starting dose of 80 mg every 3 weeks. In summary, the nonclinical pharmacology, pharmacokinetic, and toxicology data demonstrate that SRK-181 is a selective inhibitor of latent TGFß1 that does not produce the nonclinical toxicities associated with nonselective TGFß inhibition. These data support the initiation and safe conduct of a phase 1 trial with SRK-181 in patients with advanced cancer.

Selective inhibition of TGFß1 activation overcomes primary resistance to checkpoint blockade therapy by altering tumor immune landscape.

Despite breakthroughs achieved with cancer checkpoint blockade therapy (CBT), many patients do not respond to anti-programmed cell death-1 (PD-1) due to primary or acquired resistance. Human tumor profiling and preclinical studies in tumor models have recently uncovered transforming growth factor-ß (TGFß) signaling activity as a potential point of intervention to overcome primary resistance to CBT. However, the development of therapies targeting TGFß signaling has been hindered by dose-limiting cardiotoxicities, possibly due to nonselective inhibition of multiple TGFß isoforms. Analysis of mRNA expression data from The Cancer Genome Atlas revealed that TGFΒ1 is the most prevalent TGFß isoform expressed in many types of human tumors, suggesting that TGFß1 may be a key contributor to primary CBT resistance. To test whether selective TGFß1 inhibition is sufficient to overcome CBT resistance, we generated a high-affinity, fully human antibody, SRK-181, that selectively binds to latent TGFß1 and inhibits its activation. Coadministration of SRK-181-mIgG1 and an anti-PD-1 antibody in mice harboring syngeneic tumors refractory to anti-PD-1 treatment induced profound antitumor responses and survival benefit. Specific targeting of TGFß1 was also effective in tumors expressing more than one TGFß isoform. Combined SRK-181-mIgG1 and anti-PD-1 treatment resulted in increased intratumoral CD8+ T cells and decreased immunosuppressive myeloid cells. No cardiac valvulopathy was observed in a 4-week rat toxicology study with SRK-181, suggesting that selectively blocking TGFß1 activation may avoid dose-limiting toxicities previously observed with pan-TGFß inhibitors. These results establish a rationale for exploring selective TGFß1 inhibition to overcome primary resistance to CBT.

Mechanisms for kinase-mediated dimerization of the epidermal growth factor receptor.

We study a mechanism by which dimerization of the EGF receptor (EGFR) cytoplasmic domain is transmitted to the ectodomain. Therapeutic and other small molecule antagonists to the kinase domain that stabilize its active conformation, but not those that stabilize an inactive conformation, stabilize ectodomain dimerization. Inhibitor-induced dimerization requires an asymmetric kinase domain interface associated with activation. EGF and kinase inhibitors stimulate formation of identical dimer interfaces in the EGFR transmembrane domain, as shown by disulfide cross-linking. Disulfide cross-linking at an interface in domain IV in the ectodomain was also stimulated similarly; however, EGF but not inhibitors stimulated cross-linking in domain II. Inhibitors similarly induced noncovalent dimerization in nearly full-length, detergent-solubilized EGFR as shown by gel filtration. EGFR ectodomain deletion resulted in spontaneous dimerization, whereas deletion of exons 2-7, in which extracellular domains III and IV are retained, did not. In EM, kinase inhibitor-induced dimers lacked any well defined orientation between the ectodomain monomers. Fab of the therapeutic antibody cetuximab to domain III confirmed a variable position and orientation of this domain in inhibitor-induced dimers but suggested that the C termini of domain IV of the two monomers were in close proximity, consistent with dimerization in the transmembrane domains. The results provide insights into the relative energetics of intracellular and extracellular dimerization in EGFR and have significance for physiologic dimerization through the asymmetric kinase interface, bidirectional signal transmission in EGFR, and mechanism of action of therapeutics.

The C-terminal αI domain linker as a critical structural element in the conformational activation of αI integrins.

The activation of α/ß heterodimeric integrins is the result of highly coordinated rearrangements within both subunits. The molecular interactions between the two subunits, however, remain to be characterized. In this study, we use the integrin α(L)ß(2) to investigate the functional role of the C-linker polypeptide that connects the C-terminal end of the inserted (I) domain with the ß-propeller domain on the α subunit and is located at the interface with the ßI domain of the ß chain. We demonstrate that shortening of the C-linker by eight or more amino acids results in constitutively active α(L)ß(2) in which the αI domain is no longer responsive to the regulation by the ßI domain. Despite this intersubunit uncoupling, both I domains remain individually sensitive to intrasubunit conformational changes induced by allosteric modulators. Interestingly, the length and not the sequence of the C-linker appears to be critical for its functionality in α/ß intersubunit communication. Using two monoclonal antibodies (R7.1 and CBR LFA-1/1) we further demonstrate that shortening of the C-linker results in the gradual loss of combinational epitopes that require both the αI and ß-propeller domains for full reactivity. Taken together, our findings highlight the role of the C-linker as a spring-like element that allows relaxation of the αI domain in the resting state and controlled tension of the αI domain during activation, exerted by the ß chain.

Bcl-2/bcl-xL bispecific antisense treatment sensitizes breast carcinoma cells to doxorubicin, paclitaxel and cyclophosphamide.

Overexpression of the anti-apoptotic proteins bcl-2 and bcl-xL is implicated in breast cancer development, tumor progression and drug resistance. Here we describe the use of the bcl-2/bcl-xL bispecific antisense oligonucleotide 4625 to sensitize breast carcinoma cells to anti-cancer drugs routinely used in breast cancer therapy. MCF7 cells were treated with oligonucleotide 4625, doxorubicin, paclitaxel or cyclophosphamide alone, or with combinations of oligonucleotide and the anti-cancer drugs. As measured in cell viability assays, treatment with the various combinations reduced the number of viable MCF7 cells more effectively than treatment with the single drugs alone. Treatment with a sequence control oligonucleotide did not affect cell viability. All combination treatments induced apoptosis as demonstrated by the appearance of massive nuclear condensation in a high proportion of the cells. To further characterize the interaction between 4625 and doxorubicin, paclitaxel or cyclophosphamide, the median-effect method was used. In MCF7 cells all combinations resulted in potent synergistic effects over a broad range of toxicity with combination indices ranging from 0.8 to 0.1. Similarly, strong synergistic interactions between oligonucleotide 4625 and the anti-cancer drugs were also observed in cultures of the breast carcinoma cell line MDA-MB-231. Our data suggest the use of 4625 as a potent adjuvant in breast cancer chemotherapy.