Evaluation of the clinical molecule anti-human-PD-L1/IL-15 KD033 in the human-PD-1/PD-L1-expressing murine model demonstrates PD-L1 targeting of IL-15 in vivo.

KD033 is a clinical-stage immunocytokine composed of a high-affinity anti-human-PD-L1 antibody and the human IL-15/ IL-15 receptor sushi-domain complex. We have previously shown that KD033-surrogate, the anti-mouse-PD-L1/IL-15 immunocytokine, was efficacious in several syngeneic murine tumor models including those that were refractory to anti-PD-1/PD-L1 checkpoint blockers. KD033-surrogate showed better efficacy than the combination treatment of its component, anti-PD-L1 antibody with the non-targeting IL-15. KD033-surrogate was also efficacious in both low and high PD-L1-expressing tumors. In this study, we have utilized double knock-in mice expressing functional human PD-1/PD-L1 to show that the clinical molecule, KD033, reproduced the anti-tumor efficacy observed with KD033-surrogate in the syngeneic models. KD033 was equally efficacious in reducing the growth of human-PD-L1 positive (hPDL1+) and negative (hPDL1-) MC38 murine tumors. We observed similar peripheral pharmacodynamics changes in KD033-treated mice bearing either hPDL1+ or hPDL1- MC38 tumors. However, different transcriptomic profiles were observed between KD033-treated hPDL1+ and hPDL1- MC38 tumors with marked changes involving mostly downregulated genes in hPDL1- tumors in addition to the immune-related genes changes observed in both hPDL1+ and hPDL1- MC38 tumors. Cytotoxic and myeloid cell signatures were upregulated in both tumors with relatively greater increases observed in hPDL1- MC38 tumors. These effects of KD033 treatment in PD-L1 positive and negative tumors demonstrate the role of PD-L1 in targeting of IL-15 cytokine in vivo.

Uracil residues dependent on the deaminase AID in immunoglobulin gene variable and switch regions.

Activation-induced deaminase (AID) initiates diversity of immunoglobulin genes through deamination of cytosine to uracil. Two opposing models have been proposed for the deamination of DNA or RNA by AID. Although most data support DNA deamination, there is no physical evidence of uracil residues in immunoglobulin genes. Here we demonstrate their presence by determining the sensitivity of DNA to digestion with uracil DNA glycosylase (UNG) and abasic endonuclease. Using several methods of detection, we identified uracil residues in the variable and switch regions. Uracil residues were generated within 24 h of B cell stimulation, were present on both DNA strands and were found to replace mainly cytosine bases. Our data provide direct evidence for the model that AID functions by deaminating cytosine residues in DNA.

MSH2-MSH6 stimulates DNA polymerase eta, suggesting a role for A:T mutations in antibody genes.

Activation-induced cytidine deaminase deaminates cytosine to uracil (dU) in DNA, which leads to mutations at C:G basepairs in immunoglobulin genes during somatic hypermutation. The mechanism that generates mutations at A:T basepairs, however, remains unclear. It appears to require the MSH2-MSH6 mismatch repair heterodimer and DNA polymerase (pol) eta, as mutations of A:T are decreased in mice and humans lacking these proteins. Here, we demonstrate that these proteins interact physically and functionally. First, we show that MSH2-MSH6 binds to a U:G mismatch but not to other DNA intermediates produced during base excision repair of dUs, including an abasic site and a deoxyribose phosphate group. Second, MSH2 binds to pol eta in solution, and endogenous MSH2 associates with the pol in cell extracts. Third, MSH2-MSH6 stimulates the catalytic activity of pol eta in vitro. These observations suggest that the interaction between MSH2-MSH6 and DNA pol eta stimulates synthesis of mutations at bases located downstream of the initial dU lesion, including A:T pairs.

Single-Dose Anti-PD-L1/IL-15 Fusion Protein KD033 Generates Synergistic Antitumor Immunity with Robust Tumor-Immune Gene Signatures and Memory Responses.

Immunocytokines hold great potential as anticancer agents, as they use a specific antitumor antibody to deliver an immune-activating cytokine directly to the immunosuppressive tumor microenvironment (TME). We have developed a novel immunocytokine (KD033) composed of a fully human, high-affinity antiprogrammed death-ligand 1 (PD-L1) linked to the sushi-domain of the human IL-15/IL-15 receptor alpha (IL-15/IL-15Rα) complex. A murine PD-L1 cross-reactive KD033 surrogate (srKD033) and a nontargeting antibody (ntKD033) were also developed to investigate mechanism of action in murine tumor models. Efficacy analyses showed a robust antitumor effect of single-dose srKD033 in several diverse syngeneic murine tumor models. In a CT26 murine colon tumor model, single-dose srKD033 produced durable antitumor immunity as evidenced by resistance to subsequent tumor rechallenges. Mice responding to srKD033 treatment showed increased retention of PD-L1/IL-15 in the TME which likely facilitated prolonged IL-15-induced expansion of cytotoxic cells. Importantly, target-based PD-L1/IL-15 delivery via srKD033 was well-tolerated and induced significant antitumor activity in murine carcinoma models that are non- or minimally responsive to IL-15 or anti-PD-L1/PD-1 monotherapy.

A role for Msh6 but not Msh3 in somatic hypermutation and class switch recombination.

Somatic hypermutation is initiated by activation-induced cytidine deaminase (AID), and occurs in several kilobases of DNA around rearranged immunoglobulin variable (V) genes and switch (S) sites before constant genes. AID deaminates cytosine to uracil, which can produce mutations of C:G nucleotide pairs, and the mismatch repair protein Msh2 participates in generating substitutions of downstream A:T pairs. Msh2 is always found as a heterodimer with either Msh3 or Msh6, so it is important to know which one is involved. Therefore, we sequenced V and S regions from Msh3- and Msh6-deficient mice and compared mutations to those from wild-type mice. Msh6-deficient mice had fewer substitutions of A and T bases in both regions and reduced heavy chain class switching, whereas Msh3-deficient mice had normal antibody responses. This establishes a role for the Msh2-Msh6 heterodimer in hypermutation and switch recombination. When the positions of mutation were mapped, several focused peaks were found in Msh6(-/-) clones, whereas mutations were dispersed in Msh3(-/-) and wild-type clones. The peaks occurred at either G or C in WGCW motifs (W = A or T), indicating that C was mutated on both DNA strands. This suggests that AID has limited entry points into V and S regions in vivo, and subsequent mutation requires Msh2-Msh6 and DNA polymerase.

Reevaluation of the role of DNA polymerase theta in somatic hypermutation of immunoglobulin genes.

DNA polymerase theta has been implicated in the process of somatic hypermutation in immunoglobulin variable genes based on several reports of alterations in the frequency and spectra of mutations from Polq(-/-) mice. However, these studies have contrasting results on mutation frequencies and the types of nucleotide substitutions, which question the role of polymerase theta in hypermutation. DNA polymerase eta has a dominant effect on mutation and may substitute in the absence of polymerase theta to affect the pattern. Therefore, we have examined mutation in mice deficient for both polymerases theta and eta. The mutation frequencies in rearranged variable genes from Peyer's patches were similar in wild type, Polq(-/-), Polh(-/-), and Polq(-/-)Polh(-/-) mice. The types of substitutions were also similar between wild type and Polq(-/-) clones, and between Polh(-/-) and Polq(-/-)Polh(-/-) clones. Furthermore, there was no difference in heavy chain class switching in splenic B cells from the four groups of mice. These results indicate that polymerase theta does not play a significant role in the generation of somatic mutation in immunoglobulin genes.

Somatic hypermutation: subverted DNA repair.

Somatic hypermutation generates high-affinity antibodies of different isotypes that efficiently protect us against a plethora of pathogens. Recent analyses of the types of mutations produced in gene-deficient mice have indicated how DNA repair proteins are drawn into the pathway. Activation-induced cytosine deaminase begins the process by deaminating cytosine to uracil in DNA. The uracils are then recognized by the base excision repair protein uracil DNA glycosylase and by the mismatch repair proteins MutS homologue 2 and MutS homologue 6. Instead of repairing the uracils, these proteins attract low fidelity DNA polymerases, which synthesize nucleotide substitutions at an unprecedented level.

Normal hypermutation in antibody genes from congenic mice defective for DNA polymerase iota.

Several low fidelity DNA polymerases participate in generating mutations in immunoglobulin genes. Polymerase eta is clearly involved in the process by causing substitutions of A:T base pairs, whereas polymerase iota has a controversial role. Although the frequency of mutations was decreased in the BL2 cell line deficient for polymerase iota, hypermutation was normal in the 129 strain of mice, which has a natural nonsense mutation in the Poli gene. It is possible that the mice compensated for the defect over time, or that polymerase eta substituted in the absence of polymerase iota. To examine polymerase iota in a genetically defined background, we backcrossed the 129 nonsense mutation to the C57BL/6 strain for six generations. Class switch recombination and hypermutation were studied in these mice and in congenic mice doubly deficient for both polymerases iota and eta. The absence of both polymerases did not affect production of IgG1, indicating that these enzymes are not involved in switch recombination. Poli(-/-F6) mice had the same types of nucleotide substitutions in variable genes as their C57BL/6 counterparts, and mice doubly deficient for polymerases iota and eta had the same mutational spectrum as Polh-/- mice. Thus, polymerase iota did not contribute to the mutational spectra, even in the absence of polymerase eta.

Effects of biological DNA precursor pool asymmetry upon accuracy of DNA replication in vitro.

Deoxyguanosine triphosphate is underrepresented among the four common deoxyribonucleoside triphosphates (dNTPs), typically accounting for just 5-10% of the total dNTP pool. We have asked whether this pool asymmetry affects the fidelity of DNA replication, by use of an in vitro assay in which an M13 phagemid containing the Escherichia coli lacZalpha gene and an SV40 replication origin is replicated by extracts of human cells. By monitoring reversion of either a TGA or TAA codon within the lacZalpha gene, we found that replication in "biologically biased" dNTPs, representing our estimate of the concentrations in HeLa cell nuclei, is not significantly more accurate than when measured in reaction mixtures containing the four dNTPs at equimolar concentrations. However, sequence analysis of revertants revealed significantly different patterns of mispairing events leading to mutation. During replication at biased dNTP levels, mutations at the site 5' to C in the template strand for the TGA triplet were less frequent than seen in equimolar reaction mixtures, suggesting that extension from mismatches at this site is relatively slow, and proofreading efficiency high, when dGTP is the next nucleotide to be incorporated. Mismatches opposite template C, which might have been favored by the low physiological concentrations of dGTP, were not favored in our in vitro system, although one particular substitution at this site, TGA-->TTA, was strongly favored at low [dGTP]. An excess of one dNTP was found in our system to be more mutagenic than a corresponding deficiency. We also estimated dNTP concentrations in non-transformed human fibroblasts and found that in vitro replication at these levels caused significantly fewer mutations than we observed under equimolar conditions (100 microM each dNTP). This increased replication fidelity may result from increased proofreading efficiency at the lower dNTP levels; however, replication rates were decreased only slightly at these non-transformed fibroblast concentrations.

Deoxyuridine is generated preferentially in the nontranscribed strand of DNA from cells expressing activation-induced cytidine deaminase.

Activation-induced cytidine deaminase (AID) is required for somatic hypermutation and class switch recombination of Ig genes in B cells. Although AID has been shown to deaminate deoxycytidine to deoxyuridine in DNA in vitro, there is no physical evidence for increased uracils in DNA from cells expressing AID in vivo. We used several techniques to detect uracil bases in a gene that was actively transcribed in Escherichia coli cells expressing AID. Plasmid DNA containing the gene was digested with uracil-DNA glycosylase to remove uracil, and apurinic/apryimidinic endonuclease to nick the abasic site. The nicked DNA was first analyzed using alkaline gel electrophoresis, in which there was a 2-fold increase in the linear form of the plasmid after AID induction compared with plasmid from noninduced bacteria. Second, using a quantitative denaturing Southern blot technique, the gene was predominantly nicked in the nontranscribed strand compared with the transcribed strand. Third, using ligation-mediated PCR, the nicks were mapped on the nontranscribed strand and were located primarily at cytosine bases. These data present direct evidence for the presence of uracils in DNA from cells that are induced to express AID, and they are preferentially generated at cytosines in the nontranscribed strand during transcription.

Different mutation signatures in DNA polymerase eta- and MSH6-deficient mice suggest separate roles in antibody diversification.

Hypermutation in immunoglobulin genes produces a high frequency of substitutions of all four bases, which are likely generated by low-fidelity DNA polymerases. Indeed, humans deficient for DNA polymerase (pol) eta have decreased substitutions of A.T base pairs in variable and switch regions. To study the role of pol eta in a genetically tractable system, we created mice lacking pol eta. B cells from Polh-/- mice produced normal amounts of IgG, indicating that pol eta does not affect class switch recombination. Similar to their human counterparts, variable and switch regions from Polh-/- mice had fewer substitutions of A.T base pairs and correspondingly more mutations of C.G base pairs, which firmly establishes a central role for pol eta in hypermutation. Notably, the location and types of substitutions differ markedly from those in Msh6-/- clones, which also have fewer A.T mutations. The data suggest that pol eta preferentially synthesizes a repair patch on the nontranscribed strand, whereas MSH6 functions to generate the patch.